Therapeutic methods and compositions using viruses of the recombinant paramyxoviridae family

ABSTRACT

The invention relates to compositions and methods for treating a patient having a tumor in order to reduce tumor size, comprising administering to the patient a replication-competent Paramyxoviridae virus comprising two or more of a) a nucleic acid sequence encoding a heterologous polypeptide, wherein upon administration the heterologous polypeptide is detectable in a biological fluid of the patient, and detection of the heterologous polypeptide is indicative of Paramyxoviridae virus growth in the patient and reduction in tumor size; b) a recombinant F protein, H protein, or M protein of Paramyxoviridae virus that increases fusogenicity of virus with cells; c) a nucleic acid sequence encoding a cytokine; and d) a Paramyxoviridae virus that is specific for cells of the tumor.

This application claims priority to U.S. Provisional Application Ser.No. 06/155,873, filed Sep. 24, 1999, the entirety of which isincorporated by reference herein.

FIELD OF THE INVENTION

The invention relates relates to methods for production and use ofgenetically altered paramyxoviruses for the treatment of cancer.

BACKGROUND

The family Paramyxoviridae are negative strand RNA viruses known to beresponsible for a variety of human and veterinary diseases. The familycontains four genera: (i) Paramyxovirus (Sendai virus; parainfluenzavirus, types I and III; mumps virus), (ii) Morbillivirus (measles virus;rinderpest virus; phocine distemper virus; and canine distemper virus,(iii) Rubulavirus (Simian virus type V; Newcastle disease virus), and(iv) Pneumovirus (human respiratory syncytial virus; bovine respiratorysyncytial virus).

Paramyxoviridae viruses are enveloped, so possess membrane ‘spike’glycoproteins which are responsible for viral attachment to cellsurfaces via a specific receptor and for mediating virus-cell membranefusion. Subsequent to receptor binding, these viruses enter cells bydirect fusion of the viral and host cell membranes. Viruses which fuseat the target cell membrane in a pH-independent manner are activated forfusion by the receptor binding event itself triggering conformationalchanges in the envelope glycoprotein. The ability of viruses to fusedirectly with the target cell membrane is strongly associated with atendency to trigger membrane fusion between infected and neighboringuninfected cells, the visible outcome of which is the formation of largemultinucleated syncytia centering on a single infected cell.

Unmodified mumps virus administered as a tissue culture supernatant to90 patients with terminal malignancies by intratumoral, oral, rectal orintravenous inoculation, or by inhalation, and resulted in significanttumor regressions (between 50 and 100%) in 37 of the patients treated,with minor responses in a further 42 patients (Asada 1974 Cancer 341907). The activity of mumps virus was not confined to a single tumortype, but was apparent in a range of different epithelial andnonepithelial malignancies.

Newcastle disease virus, an avian Paramyxovirus, has been used to infectcancer cells which have been removed from the patient, and are thenirradiated and administered to the patient as a vaccine to elicit anantitumor immune response.

While certain Paramyxoviridae viruses have been used for cancertreatment, there is a need in the art for Paramyxoviridae viruses whichare selectively cytotoxic for tumor cells such that the virus willspread rapidly and selectively through neoplastic tissues while sparingnormal host tissues. There also is a need for a convenient and reliablemethod for monitoring the spread of the virus and the virus load in thetreated patient.

SUMMARY OF THE INVENTION

In one aspect, the invention encompasses a method of monitoring areduction in tumor size in a patient, comprising administering to apatient having a tumor a replication-competent Paramyxoviridae viruscomprising a nucleic acid sequence encoding a heterologous polypeptide,wherein upon administration the heterologous polypeptide is detectablein a biological fluid of the patient, and detection of the heterologouspolypeptide is indicative of Paramyxoviridae virus growth in the patientand reduction in tumor size. In one embodiment, the heterologouspolypeptide is biologically inactive in the patient. In anotherembodiment, the Paramyxoviridae virus comprises a chimeric gene encodinga recombinant fusion protein comprising the heterologous polypeptidefused to an endogenous polypeptide. In still another embodiment, therecombinant fusion protein comprises an amino acid linker sequencebetween the heterologous polypeptide and the endogenous polypeptide,wherein the amino acid linker sequence comprises a protease cleavagesite.

The invention also encompasses a method of increasing the fusogenicityon tumor cells of a Paramyxoviridae virus, comprising contacting tumorcells with a replication-competent Paramyxoviridae virus comprising oneor more of a recombinant F protein, H protein, or M protein of theParamyxoviridae virus that increases fusogenicity of the virus with thecells.

The invention also encompasses a method of reducing tumor size in apatient, comprising administering to a patient having a tumor areplication-competent Paramyxoviridae virus comprising one or more of arecombinant F, H, or M protein of the Paramyxoviridae virus havingincreased fusogenicity of the virus with cells of the tumor.

The invention also encompasses a method of reducing tumor size in apatient, comprising administering to a patient having a tumor areplication-competent Paramyxoviridae comprising a nucleic acid sequenceencoding a cytokine, wherein the administration results in reduced tumorsize.

The invention also encompasses a method of reducing tumor size in apatient, comprising administering to a patient having a tumor aParamyxoviridae virus that is specific for cells of the tumor.Preferably, the Paramyxoviridae virus comprises a viral surface ligandthat specifically binds to a receptor on a tumor cell. In oneembodiment, the ligand is fused via an intervening amino acid linker toa Paramyxoviridae virus surface protein to form a ligand/virusrecombinant protein such that the fusion protein specifically binds tothe receptor on the tumor cell. In another embodiment, the amino acidlinker of the fusion protein comprises a protease cleavage site for aprotease produced by the tumor cell, such that cleavage of the cleavagesite by the protease produced by the tumor cell permits infection of thetumor cell by the Paramyxoviridae virus.

Preferably, the virus surface protein is one of F protein or H protein.

Preferably, the ligand is a single chain antibody specific forcarcinoembryonic antigen and the tumor cell receptor is carcinoembryonicantigen.

Preferably, the furin cleavage sequence of the Paramyxoviridae virus Fprotein is removed and replaced with a cleavage sequence of a proteaseproduced by the tumor cell.

The invention also encompasses a method of producing a recombinantParamyxoviridae virus comprising, in order, the steps of: 1)transfecting a eukaryotic cell line stably expressing T7 RNA polymerasewith an infectious Paramyxoviridae viral genomic cDNA under the controlof a T7 promoter; 2) infecting the transfected cells of step (1) with ahelper virus expressing a selectable trait, and Paramyxoviridae viral N,P and L proteins; 3) contacting the infected, transfected cells of step(2) with cells that permit Paramyxoviridae virus infection andreplication, under conditions permitting the infection and replication;4) selecting syncytia formed on the cells that permit Paramyxoviridaevirus infection and replication; 5) screening for and isolating theParamyxoviridae lacking helper viral genetic material based upon thepresence or absence of the selectable trait of the helper virus; and 6)expanding the Paramyxoviridae virus lacking helper viral geneticmaterial to produce the recombinant Paramyxoviridae virus. In oneembodiment, the selectable trait is deletion of F protein. In anotherembodiment, the selectable trait is expression of an F protein that iscleavable by a protease other than furin. In another embodiment, theselectable trait is expression of GFP.

The invention also encompasses a kit for treatment of a patient having atumor, the kit comprising a replication-competent Paramyxoviridae viruscomprising one or more of: a) a nucleic acid sequence encoding aheterologous polypeptide, wherein upon the administration theheterologous polypeptide is detectable in a biological fluid of thepatient, and detection of the heterologous polypeptide is indicative ofParamyxoviridae virus growth in the patient and reduction in tumor size;b) a recombinant F protein, H protein, or M protein of theParamyxoviridae virus that increases fusogenicity of the virus with thecells; c) a nucleic acid sequence encoding a cytokine; and d) aParamyxoviridae virus that is specific for cells of the tumor.

The invention also encompasses a method of treating a patient having atumor in order to reduce tumor size, comprising administering to thepatient a replication-competent Paramyxoviridae virus comprising two ormore of: a) a nucleic acid sequence encoding a heterologous polypeptide,wherein upon the administration the heterologous polypeptide isdetectable in a biological fluid of the patient, and detection of theheterologous polypeptide is indicative of Paramyxoviridae virus growthin the patient and reduction in tumor size; b) a recombinant F protein,H protein, or M protein of the Paramyxoviridae virus that increasesfusogenicity of the virus with the cells; c) a nucleic acid sequenceencoding a cytokine; and d) a Paramyxoviridae virus that is specific forcells of the tumor.

In one embodiment, in any of the inventive methods and compositions, theParamyxoviridae virus is selected from the group consisting ofParamyxovirus, Morbillivirus, Rubulavirus and Pneumovirus. In anotherembodiment, the Paramyxovirus is one of mumps virus, parainfluenzaviruses type I or III, and Sendai virus. In still another embodiment,the Morbillivirus is one of measles virus, rinderpest virus, phocinedistemper virus, and canine distemper virus. In a further embodiment,the Rubulavirus is one of Newcastle disease virus and Simian virus V. Instill a further embodiment, the Pneumovirus is one of human respiratorysyncytial virus and bovine respiratory syncytial virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the plasmid encoding MV H proteinfused to a C peptide heterologous polypeptide that is either cleavable(pCGH FurCP) or non-cleavable (PCGH G4S CP).

FIG. 2 shows a schematic diagram of the construction of the plasmidp(+)MPGFPV from the plasmid p(+)MPCATV.

FIG. 3 shows a schematic diagram of the intermediate plasmid constructp(+)MPIrGFPV.

FIG. 4 shows a schematic diagram of the plasmid p(+)MIrGFPNV.

FIG. 5. Generation and characterization of recombinant MV with targetingspecificity for CD38. Schematic representation of the genome ofrecombinant MV displaying single chain antibodies as C-terminal fusionsto H glycoprotein. The scFvs are linked to H glycoportein via a FactorXa protease cleavage (IEGR) site.

FIG. 6A-D. recombinant MV displaying anti-CD38 antibody efficeintlyinfects CD38-expressing CH) cells. The CHO0CD38 target cells areinfected with (A)-anti-CD38 MV or (B) anti-CD52 MV. The number ofinfectious centers (synctia with >20 nuclei) were counted and expressedas syncytial forming units/ml of virus. (C) Virus output from CHO-CD38and (D) expressed as syncytial forming units/ml of virus. (C) Virusoutput from CHO-CD38 and (D) Vero cells, 48 hours following infectionwih untreated (black histogram_or Factor Xa protease treated (greyhistogram) ti-CD38 or anti-CD52 viruses.

FIG. 7. Immunoblot to demostrate that the recombinant MV expresseshybrid glycoproteins.

FIGS. 8A-B: (A) Schematic Diagram Showing The Relative Positions Of MVH, Spacer Incorporating Factor Xa Cleavage Site And Appended Scab. TheLinker Sequences Separating V_(H) And V_(L) In The Three Scab Forms,Indicated As 0, S And L, Are Also Given. (B) HαCEA Are Expressed At TheExpected Molecular Weight. Western Blot Analysis Using An αFlag Ab OfVero Cells Transfected With The Indicated H Constructs Or MockTransfected. Numbers Correspond To MW In Thousands.

FIGS. 9A-B: (A) MV-HXL infects and induces cell-cell fusion inCD46-negative, CEA-positive cells. Representative fields of view werephotographed 72 hours post-infection with MV, MV-HXL (MOI 3) or novirus. (B) MV-HXL replicates in CD46-negative, CEA-positive cells.Infectivities were quantified by TCID₅₀ titration using the indicatedcell lines as targets.

FIGS. 10A-D: (A) HXL protein induces syncytia formation in CD46-positivecells. Syncytia were scored 48 hours post-co-transfection with MV F andthe indicated H constructs. (B) Expression levels of CD46 and CEA on allcell lines used in this study. Grey shading, no primary Ab as negativecontrol; thin line, CEA; thick line, CD46. (C) and (D) HXL proteininduces syncytia formation in CD46-negative, CEA-positive cells.Syncytia were scored (C) and photographed (D) 48 hourspost-co-transfection with F and the indicated H constructs. For syncytiascoring, H and HXL on Vero cells were not countable as >90% of cellswere in syncytia. Black arrows indicate isolated syncytia.

FIG. 11A-C: (A) MV-HXL expresses an H protein of the expected molecularweight. Western blot analysis of purified particles with an MV-specificantiserum, numbers refer to MW in thousands. (B) Cleavage of thedisplayed scAb from MV-HXL by Factor Xa protease treatment. Western blotanalysis of purified particles with an αMV serum following Factor Xatreatment (10 μg/ml, 2 hours, 23° C.), numbers refer to MW in thousands.(C) MV-HXL binds the surface of CD46-negative, CEA-positive cells. Boundvirus (MOI 3) was detected by FACS analysis using mAb I29. Grey shading,no virus; thin line, MV; thick line, MV-HXL.

FIG. 12: MV-HXL infectivity for MC38-CEA cells is inhibited by cleavageof the displayed scAb or antibody preadsorption of cell surface CEA.Cells were infected with MV or MV-HXL (MOI 1), untreated or pretreatedwith 10 μg/ml Factor Xa. Also, cells were pretreated with 10 μg/ml αCEACOL1 Ab, then infected with MV or MV-HXL (MOI 1). Virus released fromthe cells by freeze/thaw 72 hours post-infection was quantified byTCID₅₀ titration on Vero cells.

FIG. 13A-C: (A) HαCEA proteins are as stable as unmodified MV H. Pulsechase analysis of Vero cells transfected with the indicated Hconstructs. Antigenic material was precipitated after the indicatedchase times in minutes. (B) HXL protein dimerizes with itself and withunmodified H. Vero cells co-transfected with the indicated constructswere radiolabelled and lysed. Material immunoprecipitated with αFlag mAbwas dissolved under non-reducing conditions, numbers refer to MW inthousands. (C) HX0, HXS and HXL are localized at the cell surface. FACSanalysis using mAb I29 of MC38 cells transfected with the indicated Hconstructs. Grey shading, no primary Ab.

FIGS. 14A-E. Genomic structure, protein composition and replication ofrecombinant MV. (A) Plasmid p(+)MV-NSe coding for the MV antigenome(top), PacI-SpeI fragments used for subcloning (center), and amino acidsequences (one letter code) of the junctions between the H proteinectodomain and the specificity domains (bottom). Coding regions of thesix MV cistrons are represented by solid black boxes, the transmembranesegment of the F and H proteins by an horizontally lined box, the FXacleavage site and the flexible linker by a hatched box and the ligand bya gray box. Arginine residues in brackets have been deleted. For detailssee text. (B) Protein composition of recombinant viruses. Viralparticles were purified by centrifugation through a 20% sucrose layeronto a 60% sucrose cushion and subsequent pelleting. The viruses weretitrated, 5000 pfu were subjected to lysis, and proteins were separatedby SDS-PAGE. For immunodetection a MV-specific antiserum was used. (C)FXa protease sensitivity of hybrid H proteins. Purified viral particles(5000 pfu) were lysed and incubated for 1 hour without (−) or with (+)FXa protease at room temperature. Proteins were separated by SDS-PAGEand detected with a H-specific antiserum. (D) FACS analysis of CD46(interrupted line), IGF1r (dotted line) and EGFr (continuous line)expression on Vero cells. Vertical axis: cell number. Horizontal axis:fluorescence intensity. The greyed profile represents incubation of thecells without the primary antibody. (E) Time course of released (rightpanel) and cell-associated (left panel) virus production in Vero cellsinfected with parental MV (diamonds), MV-H/XhEGF (squares) andMV-H/XhIGF1 (triangles). Cells were infected at a m.o.i. of 3 andincubated at 32° C. for the times indicated. Viral titers weredetermined by 50% end-point dilution. Indicated values are averages ofthree experiments.

FIGS. 15A-B. Binding of recombinant viral particles to (A) immobilizedhEGF- and hIGF1-specific antibodies and to (B) rodent cells expressing atarget receptor. (A) ELISA plates were coated either with anti-hEGF oranti-hIGF1 monoclonal antibodies. After blocking, the plates wereincubated with different dilutions of MV-H/XhEGF and MV-H/XhIGF1. Boundvirus was detected using an H-specific antiserum. (B) Expression levelsof hEGFr in CHO-hEGFr cells (top left) and of hIGF1r in 3T3-hIGF1 cells(top right); cells incubated only with the secondary antibody showedbackground level of fluorence (mean 3.9 versus 466.5 for CHO-hEGFr cellsand 5.7 versus 250.8 for 3T3-hIGF1 cells, data not shown). Binding ofMV-H/XhEGF to CHO-hEGFr cells (bottom left) and of MV-H/XhIGF1 to3T3-hIGF1r cells (bottom right). Thick lines: incubations with therecombinant viruses. Thin lines: incubation with MV. Grey areas: controlincubations without virus. Vertical axis: cells counted. Horizontalaxis: fluorescence intensity.

FIGS. 16A-F. Infection of CHO-hEGFr cells, CHO-hEGFr.tr cells (G and H)and CHO cells (I and J) with MV^(green)-H/XhEGF (A-D and G-J) or MV (Eand F). Cells were infected at a m.o.i. of 1 and monitored byfluorescent- or phase-contrast microscopy 24 (A), 48 (B) or 72 (C-J)hours after infection.

FIG. 17A-D. Infection of 3T3-hIGF1r cells with MV^(green)-H/XhIGF1r (Aand B) or MV^(green) (C and D). Cells were infected at a m.o.i. of 3 andinfection was monitored by fluorescent-(A and C) or phase-contrast (Band D) microscopy 24 hours after infection. FIG. 18A-B. Competition ofviral entry by soluble receptors (A) and effect of proteolytic cleavageof the specificity domain on entry (B). (A) Competition of viralinfections by soluble EGF (sEGF, top panel) or soluble IGF1 (sIGF1,bottom panel). CHO-hEGFr and Vero cells pretreated or not with sEGF wereinfected with MV^(green) or MV^(green)-H/XhEGF at an m.o.i. of 0.3.3T3-hIGFr and Vero cells pretreated or not with sIGF1 were infected withMV^(green) and MV^(green)-H/XhIGF1 at an m.o.i. of 0.1. The percentageof infected cells was determined at 24 hours p.i. by counting GFPexpressing cells in a standard area, and normalized to the number of GFPexpressing cells in infections without added soluble receptor. Resultsobtained with MV Edmonston on Vero cells are indicated with graycolumns. Results obtained with recombinant MV on Vero cells with whitecolumns, and on rodent cells with black columns. (B) Effects of thetreatment of recombinant viruses with FXa protease prior to infection.Vero, CHO-hEGFr or 3T3-hIGF1 r cells were infected withMV^(green)-H/XhEGF or MV^(green)-H/XhIGF1 (10⁴ pfu/well), respectively,pretreated with 0, 5 or 50 μg/ml FXa protease. The number of GFPexpressing cells was determined 42 hours p.i. and normalized tountreated virus.

FIG. 19 shows that a heterologous polypeptide, CEA, can be used to trackthe replication of a therapeutic measles virus according to oneembodiment of the invention.

DESCRIPTION

The invention is based upon the discovery that recombinantParamyxoviridae viruses, containing modifications, either singly, or incombination, are advantageous for cancer therapy, in terms of safety,efficacy, and/or ability to monitor. The recombinant Paramyxoviridaeviruses according to the invention enhance the therapeutic effect of aParamyxoviridae virus relative to its unmodified counterpart.

Definitions

In order to more clearly and concisely describe and point out thesubject matter of the claimed invention, the following definitions areprovided for specific terms which are used in the following writtendescription and the appended claims.

As used herein, the term “monitoring” refers to a process of determiningthe amount of viral replication or gene expression occurring in a hostcell, tissue or organism.

As used herein, “Paramyxoviridae virus” refers to a negative strand RNAvirus of the family Paramyxoviridae. The family Paramyxoviridae containsthree genera: (i) Paramyxovirus (Sendai virus; parainfluenza virus,types I, II, III and IV; mumps virus; simian virus type V; Newcastledisease virus), (ii) Morbillivirus (measles virus; rinderpest virus;phocine distemper virus; and canine distemper virus, and (iii)Pneumovirus (respiratory syncytial virus, etc.).

As used herein, “reduction in tumor size” refers to any decrease in thesize of a tumor following administration of a paramyxovirus relative tothe size of the tumor prior to administration of the virus. A tumor maybe considered to be reduced in size if it is at least 10% smaller, 25%,50% up to 100% or no tumor remaining as measured by determination oftumor mass or size, either measured directly in vivo (i.e., bymeasurement of tumors directly accessible to physical measurement, suchas by calipers) or by measurement of the size of an image of the tumorproduced, for example by X-ray or magnetic resonance imaging.

By “patient” is meant an organism which is a donor or recipient ofexplanted cells or the cells themselves. “Patient” also refers to anorganism to which viruses of the invention can be administered.Preferably, a patient is a mammal, e.g., a human, primate or a rodent.

As used herein, the term “replication-competent” refers to a virus thatis fully capable of infecting and replicating in a host cell. Areplication-competent virus requires no additional viral functionssupplied by, for example, a helper virus or a plasmid expressionconstruct encoding such additional functions.

As used herein, the term “conditions permitting the infection andreplication” refers to the collection attributes of a cell or itssurroundings that allow a given virus to infect (i.e., to insert itsgenetic material into the host, express proteins from the geneticmaterial and replicate its genetic material), and assemble newinfectious particles in, a host cell.

As used herein, the term “detectable” refers to a property of apolypeptide that allows one to determine the presence and/or amount ofthe polypeptide in a biological sample. The meaning of the term“detectable” is intended to encompasses detection of activities, forexample, enzyme activity or fluorescence activity possessed by thepolypeptide, in addition to detection of the polypeptide by other means,for example, immunoassay or mass spectroscopy.

As used herein, the term “biological fluid” refers to any extracellularbodily fluid, including but not limited to blood, urine, saliva,interstitial fluid, lymph, and cerebrospinal fluid.

As used herein, “Paramyxoviridae virus growth” refers to growth orreplication of a virus of the Paramyxoviridae family as measured byviral propagation by successive rounds of infection and replicationoccurring in a host organism, or as measured by virus titer, or asmeasures by detection of a heterologous polypeptide, or as measured by areduction in tumor size.

As used herein, the term “selecting syncytia” refers to the process ofphysically isolating or harvesting syncytia from a monolayer cultureinfected with a Paramyxoviridae virus in order to further propagate theparticular form of the virus contained within a particular syncytium.

As used herein, the terms “wild-type” or “wild-type virus” refer to thecharacteristics of a virus of the family Paramyxoviridae as it is foundin nature. The terms may be applied to any strain of a virus of thefamily Paramyxoviridae that occurs in nature, such as the Edmonston Bstrain of measles virus or other non-genetically engineered virus andcan include point mutations.

As used herein, the term “recombinant” refers to a virus or polypeptidewhich is altered by genetic engineering, by modification or manipulationof the genetic material encoding that polypeptide or found in the virussuch that it is not identical to the naturally occurring virus, or anaturally occuring variant of the virus, or polypeptide.

As used herein, the term “screening for and isolating” refers to aprocess whereby a particular virus is first identified on the basis of aparticular characteristic or selectable trait, and then isolated orisolated and expanded from among a population of viruses.

As used herein, the term “based on the presence or absence of theselectable trait” refers to a selection process whereby a virusexhibiting a particular characteristic is selected from among apopulation of viruses not exhibiting that characteristic on the basis ofselection for or against that characteristic. For example, if one wishesto select viruses that do not contain a fluorescent marker, one willselect a virus based on the absence of fluorescence in infected cells.If, on the other hand, one wishes to select virus that has a fluorescentmarker, one will select such a virus based on the presence offluorescence in infected cells.

As used herein, the term “expanding” refers to the process whereby aparticular virus is propagated in host cells in order to increase theavailable number of copies of that particular virus, preferably by atleast 2-fold, more preferably by 5-10-fold, or even by as much as50-100-fold relative to unexpanded cells.

As used herein, the term “heterologous polypeptide” refers to apolypeptide not found in nature in the Paramyxovirus strain that ismodified to contain a sequence encoding the polypeptide.

As used herein, “biologically inactive” refers to the property of apolypeptide whereby the polypeptide does not influence the propagationor cytotoxicity of a Paramyxoviridae virus.

As used herein, “endogenous polypeptide” refers to a polypeptide thatoccurs in nature in the strain of Paramyxoviridae virus into which aheterologous polypeptide is inserted.

As used herein, the term “expression of GFP” refers to the production ofgreen fluorescent protein, or a portion thereof retaining fluorescenceactivity, wherein such production is encoded by a virus that infects acell.

As used herein, the term “amino acid linker sequence” refers to asequence of amino acids that physically links and is located between twopolypeptides or polypeptide regions. A linker is from 6 amino acids to50 amino acids or from 10 amino acids to 30 amino acids and ispreferably 15 amino acisd.

A “protease cleavage site” useful in the invention is a contiguoussequence of amino acids connected by peptide bonds which contains (i) apair of amino acids which is connected by a peptide bond that ishydrolyzed by a particular protease.

As used herein, the term “deletion of F protein” refers to a helpervirus that does not express an F protein capable of cooperating with Hprotein to induce virus-cell or cell-cell fusion.

As used herein, the term “F protein that is cleavable by a proteaseother than furin” refers to a Paramyxoviridae virus F protein requiringproteolytic cleavage by an enzyme other than furin for the ability topromote virus-cell or cell-cell fusion. Furin is a protease that cleavesF proteins of Paramyxoviridae viruses; therefore a method of selectiondependent on proteolytic cleavage of F protein must exclude expressionof furin-cleavable F protein by a helper virus. A protease used inselection against viruses containing helper viral genomic material ispreferably one that may be added to or removed from cell culture medium.

As used herein, the term “increasing the fusogenicity” refers to achange in the rate or degree to which a particular modified virusinduces cell-cell fusion in an infected host.

As used herein, the term “cytokine” refers to a protein that stimulatesthe immune response in a patient.

As used herein, the term “stimulates the immune response” means that aselected response against the tumor or antigens of the tumor is faster,more efficient, more easily induced, and/or greater in magnituderelative to the absence of administration of a virus according to theinvention. The selected immune response can be stimulation or activationof a selected immune response, e.g., selective enhancement of an immuneresponse to the tumor cells.

As used herein, the term “specific for” refers to a Paramyxoviridaevirus that infects only host cells exhibiting a particularcharacteristic, such as a particular cell surface antigen orpolypeptide, or refers to a specific interaction between a ligand andits cognate receptor to the exclusion of other interactions involvingother ligands and receptors.

As used herein, the term “selectable trait” refers to a characteristicthat allows one to retain or remove cells or viruses possessing andexhibiting that trait on the basis of that trait. A selectable trait mayallow positive selection, wherein cells or viruses exhibiting that traitare selectively retained. Alternatively, a selectable trait may be anegative selectable trait, whereby cells or viruses exhibiting thattrait are deleted or removed from a population.

Recombinant Paramyxoviridae Viruses According to the Invention

A. Modifications Allowing Monitoring of the Course of Infection and/orTreatment

It is important to monitor the expression of a therapeutic virus duringthe course of treatment. In one embodiment, the invention provides agenetically engineered Paramyxoviridae viruses which encodes aheterologous polypeptide. The heterologous polypeptide is released frominfected cells into a body fluid where its concentration can bemonitored, making it a marker which provides an index of the totalnumber of virus producing cells in the body.

1. Heterologous Polypeptides.

A heterologous polypeptide useful according to the invention is anypolypeptide that is selected according any, or all, of the followingcriteria: (1) It is preferably small (e.g., having a molecular weightbelow 10 kilodaltons (kD)) and soluble in biological fluids so as toallow rapid equilibration between interstitial and intravascular fluidspaces in the body; (2) there should be a convenient, sensitive,specific, and accurate assay available for detection of the polypeptide;(3) the background level of expression of the heterologous polypeptideshould be negligible in the biological fluid being tested or thereshould be a reliable method to discount background levels wheninterpreting an assay; (4) The biodistribution, metabolism and excretionof the peptide should be well characterized and its plasma half-lifeshould be known; (5) the heterologous polypeptide should be relativelynonimmunogenic so that its half-life will not be influenced by a humanimmune response (e.g., the polypeptide is not be cleared from the bodybefore it is monitored); and (6) the heterologous polypeptide preferablyeither lacks biological activity or only has a biological activity thatis advantageous to the outcome of the therapy.

A heterologous polypeptide is used to monitor viral growth and isreadily detectable in biological fluid samples. Preferably, theheterologous polypeptide is non-immunogenic, meaning that it is notlikely to produce any significant immune response in the host organismundergoing gene therapy with the heterologous polypeptide. Theheterologous polypeptide is also preferably non-functional, which meansthat it lacks any significant known biological activity other than thatrequired to serve its use as a heterologous polypeptide (i.e., anactivity that is detectable).

Both the properties of non-immunogenicity and non-functionality aremerely intended to improve the performance of the heterologouspolypeptide by preventing undesirable side effects in the host organism.The requirements of non-immunogenicity and non-functionality are notintended to be absolute, and it is understood that a heterologouspolypeptide of the invention may possess an insignificant remnant ofbiological activity or immunogenicity in the host organism and maypossess significant immunogenicity or biological activity in an organismother than the host organism.

i. Naturally Occurring Heterologous Polypeptides as Markers

a. Cleavage Products as Markers

Naturally occurring polypeptides with very low background levels ofexpression are ideally suited to be heterologous polypeptides since theyare usually non-immunogenic. In one embodiment, a biologically inactivepolypeptide is used which is a cleavage product of a prohormoneprocessing reaction. In one embodiment, the heterologous polypeptide isC-peptide, which is the cleavage product of the prohormone proinsulin,which is found in plasma at levels of C-peptide, 170-900 pmol/l(proinsulin being found at 3-20 pmol/l, fasting insulin, being found at43-186 pmol/l). Both endogenous insulin and C-peptide levels can besuppressed using somatostatin for improved background correction, andC-peptide peripheral kinetics have been extensively studied in bothnormal volunteers and diabetic patients. Patients with type I diabetesdo not synthesize insulin and therefore have zero background levels ofC-peptide (K. S. Polonsky et al., J. Clin. Invest. 77: 98-105 (1986)).An assay for quantifying C-peptide in human blood is described in P. C.Kao et al., Ann. Clin. Lab. Science 22: 307-316 (1992), the entirety ofwhich is incorporated by reference herein.

Other polypeptide cleavage products encompassed within the scope of theinvention, include, but are not limited to, proopiomelanocortin,preproenkephalin, preprodynorphin, preprovasopressin, preprooxytocin,preprocorticotrophin releasing factor, preprogrowth hormone releasingfactor, preprosomatostatin, preproglucagon, preprogastrin,preprocalcitonin, preproepidermal growth factor, preprobradykinin,preprotachykinin, preangiotensinogen, preprovasoactive intestinalpeptide and other peptide hormone precursors (J. Douglass et al., Ann.Rev. Biochem. 53: 665-715 (1984); D. H. Lynch and S. H. Snyder, Ann.Rev. Biochem. 55: 773-799 (1986); J. C. Hutton, Diabetalogia 37 (suppl.2): S48-S56 (1994)).

b. Activation Peptides as Markers

In another embodiment of the invention, the activation peptides releasedduring the proteolytic processing of zymogens to generate active enzymes(e.g., proteases) are used to provide heterologous polypeptides. In oneembodiment, the activation peptide released by a pancreatic proenzymeduring its trypsin-induced activation is used as a heterologous peptide.Most such peptides are small (less than 1 kDa) and rapidly excreted inthe urine, enabling urine tests to be performed as a quicksemi-quantitative assay for viral expression, such as the assaydescribed in Mithofer, et al., Anal. Biochem. 230: 348-350 (1995).

In another embodiment, the activation peptide of procarboxypeptidase Bwhich is about about 10 kD (K. K. Yamamoto et al., J. Biol. Chem. 267:2575-2581 (1992)) is used as a heterologous protein, because it can bereadily measured in serum or urine (see, e.g., Appelros, et al., Gut 42:97-102 (1998)). In still another embodiment, the activation peptidesderived from enzymatic cascade reactions (e.g., such as blood clottingare used) are used as heterologous proteins since they can be assayed beroutine techniques (see, e.g., Philippou, Brit. J. Haem. 90: 432-437(1995)).

C. Inactivation Peptides as Markers

In a further embodiment, heterologous polypeptides are used which arethe fragments of hormones, proteases, or other biological molecules thathave be proteolytically inactivated. In this embodiment, peptides areselected which are relatively non-immunogenic and non-biologicallyfunctional, as discussed above. Examples of such polypeptides includecomplement peptides C3b (iC3b), C4c and C4d (see, e.g., U.S. Pat. No.5,981,481), the peptide fragments of endorphins, enkephalins, or atrialnatriuretic peptide (ANP) (see, e.g., U.S. Pat. Nos. 5,731,306 and5,714,347), and the inactivation peptides of thyrotropin-releasinghormone (TRH), substance P, neurotensin, and vasopressin (see, e.g.,EP-A 468469), and the like.

d. Tumor Antigens as Markers

Convenient, sensitive assays have been developed to detect tumorantigens in the blood, and therefore, in one embodiment, theheterologous polypeptide is a tumor antigen which is produced inexcessive amounts by a specific tumor subtype not found in the patientbeing treated. In one embodiment of the invention, the antigen isselected from the group consisting of CA125 (specific for ovariancancer), alphafetoprotein (AFP, specific for liver cancer),carcinoembryonic antigen (CEA, specific for colon cancer), intactmonoclonal immunoglobulin or light chain fragments (specific formyeloma), and the beta subunit of human chorionic gonadotrophin (HCG,specific for germ cell tumors).

CEA is an example of a large polypeptide (its molecular weight isbetween 175 kD-200 kD) which can be used successfully as a heterologousmarker polypeptide. Although the size of the polypeptide prevents itfrom being filtered by the kidney and excreted in the urine, CEA can bemeasured in the blood where its half life is approximately 5 days.Because CEA accumulates over time and equilibrates with theextracellular fluid compartment, total body CEA production can becalculated from a knowledge of its half-life and total bodyextracellular fluid volume. In the early stages after infection with aparamyxo virus bearing a CEA heterologous peptide, CEA accumulates inextracellular fluid at the site of release and diffuses only slowly intoblood vessels. However, as shown in FIG. 19, taking this lag time intoaccount the appearance of CEA correlates well with the replicativespread of virus in a tumor and tumor regression.

e. Inactive Variants as Markers

In another embodiment of the invention, heterologous polypeptides areinactive variants of naturally occurring peptides and are detected usingvariant specific antibodies. Methods of generating variant antibodiesare well known in the art and are dislosed in, U.S. Pat. No. 6,077,519,U.S. Pat. No. 6,054,273, U.S. Pat. No. 6,022,683, and U.S. Pat. No.5,773,222, the entireties of which are incorporated herein by reference.

In one embodiment, the fragment or sequence variant derived from theactive portion of any polypeptide hormone is used as a marker.Polypeptide hormones encompassed within the scipe of the invention,include, but are not limited to, gastrin, renin, prolactin,adrenocorticotrophic hormone, parathyroid hormone, parathyroid hormonerelated polypeptide, arginine vasopressin, beta endorphin, atrialnaturetic factor, calcitonin, insulin, insulin-like growth factor,glucagon, osteocalcin, erythropoietin, thrombopoietin, human growthhormone, and others.

Analogous hormones from other non-human species are also a source ofpeptide sequences which can be adopted or modified to serve as a markerpolypeptide in the invention. Many of the commercially available assaysfor such hormones have the power to detect biologically inactive,truncated, or point-mutated variants of the natural polypeptide. Forexample, deletion of the first six N-terminal amino acids of parathyroidhormone (an 84 residue polypeptide whose normal blood level is 1.0-5.2pmol/l) destroys biological activity, but the truncated molecule isstill detectable using a standard immunoassay.

An unprocessible variant of a naturally occurring precursor polypeptidecan also serve as a heterologous marker polypeptide. For example,proinsulin is processed to insulin and C-peptide by cellular proteasesthat cleave the junctions between the C-peptide and the A and B chains.Processing can be inhibited by mutation of these cleavage sites, suchthat the inactive, point-mutated proinsulin (normal level 3-20 pmol/l)will be released from the cell and detected in the blood. Similarly,variants of naturally occurring polypeptides with prolonged circulatinghalf-lives can be used as marker polypeptides. Peptide elimination canbe reduced by modifications that increase size or anionic charge(reduced glomerular filtration), by mutations in the recognition sitesfor inactivating proteases, and by mutations that lead to loss ofreceptor binding activity (reduced receptor-mediated clearance) (C.McMartin, Biochem. Soc. Trans. 17: 931-934 (1989)).

ii. Synthetic Non-Human Peptide as Markers

A fully synthetic or a non-human peptide is also useful as aheterologous marker polypeptide. Such peptides have been used to monitorprotein expression and to track synthetic proteins during purification(e.g., FLAG tag, myc tag, strep tag). Similar peptides can be designedwhich lack immunogenicity in humans. To design such a peptide, one mayuse a peptide derived from a protein not known to be immunogenic or usea peptide derived from a self protein not known for autoimmunity. Asused herein, “relatively non-immunogenic” refers to a protein or peptidethat does not elicit a deleterious immune response in a majority oftreated individuals, that is an immune response that compromises thepatients' health or that interferes with detection of the heterologouspolypeptide or the reduction in tumor size that is achieved in theabsence of the marker polypeptide. If there is an immune response to aselected peptide, it is preferred that the response is cell-mediated,rather than antibody-mediated, since the primary concern for the markerpeptide is the half-life of the peptide.

2. Inserting Marker Polypeptide Encoding Sequences Within the ViralGenome

A sequence encoding a marker polypeptide according to the invention maybe inserted into the Paramyxoviridae virus genome using a plasmidcontaining the Paramyxoviridae virus genome coding sequences, such asp(+)MV (Radecke et al., 1995, EMBO J. 14: 5773; EMBL Accession No.Z66517), and standard molecular cloning techniques (Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, 1989, (ColdSpring Harbor Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1988,Current Protocols in Molecular Biology, (John Wiley and Sons, Inc., NewYork); Ausubel et al., 1992, Short Protocols in Molecular Biology, (JohnWiley and Sons, Inc., New York).

Restriction enzyme cleavage sites occurring between the coding sequencesof the MV genome include, but are not limited to BsiWI, SpeI and AatII(between the P and M coding units), NarI (between the M and F codingunits), PacI (between the F and H coding units) and SpeI between the Hand L coding units of the p(+)MV plasmid. Sequences may be engineered byone of skill in the art to be compatible with insertion into any ofthese cloning sites. Alternatively, any of these sites may be modifiedby insertion or deletion of sequences to generate other restrictionsites useful for insertion of marker polypeptide coding sequences. Inaddition to modification of the Paramyxoviridae virus genome intergenicsequences in the plasmid to generate new restriction sites, regulatoryelements may be introduced to the intergenic region to aid in theexpression of the inserted sequences. For example, translation stopcodons may be introduced upstream of the new restriction sites, as maybe promoter sequences to drive expression of the marker polypeptide.

3. Expression of Heterologous Polypeptides as Protease Cleavable Fusions

i. Protease Sensitive Linkers

The heterologous marker polypeptide can be introduced into the viralgenome either as a protease-cleavable fusion to a virally encodedprotein or as an independent expression unit within the viral genome. Inthis way, a fixed stoichiometric relationship will exist between theexpression of the viral gene products and the marker peptide. In oneembodiment of the invention, the heterologous protein is fused to viralprotein by an amino acid acid linker sequence comprising a proteasecleavage site. A protease cleavage site may include one or more aminoacids on either side of the peptide bond to be hydrolyzed, to which thecatalytic site of the protease also binds (Schecter and Berger, Biochem.Biophys. Res. Commun. 27, 157-62, 1967), or the recognition site andcleavage site on the protease substrate may be two different sites thatare separated by one or more (e.g., two to four amino acids).

The specific sequence of amino acids in the protease cleavage sitedepends on the catalytic mechanism of the protease, which is defined bythe nature of the functional group at the protease's active site, asdiscussed above. For example, trypsin hydrolyzes peptide bonds whosecarbonyl function is donated by either a lysine or an arginine residue,regardless of the length or amino acid sequence of the polypeptidechain. Factor Xa, however, recognizes the specific sequenceIle-Glu-Gly-Arg, but hydrolyzes peptide bonds between Arg-Thr andArg-Ile. Thus, a protease cleavage site comprises at least 2, 3, 4, 5,6, 7, 8, 9, or 10 or more amino acids. Optionally, additional aminoacids can be present at the N-terminus and/or C-terminus of therecognition site.

The invention permits a great deal of flexibility and discretion interms of the choice of the protease cleavable linker peptide. Theprotease specificity of the linker is determined by the amino acidsequence of the linker. Specific amino acid sequences can be selected inorder to determine which protease will cleave the linker; this is animportant indication of the location of cleavage within the cell orfollowing secretion from the cell and can have a major effect on therelease of the marker polypeptide and its transportation through thebody of the patient. The size in the linker can generally be varied. Inone embodiment, the linker sequence is between 3 and 50 amino acids inlength. More preferably the linker is between 5 and 25 amino acids inlength, and most preferably the linker is between 8 and 15 amino acidsin length.

In one embodiment, the amino acid linker is linked by a peptide bond tothe C-terminus of the N-terminal polypeptide of the recombinant fusionprotein and via a peptide bond to the N-terminus of the C-terminalpolypeptide of the recombinant fusion protein. In one embodiment, theN-terminal polypeptide of the fusion protein is heterologous, and thusthe C-terminal polypeptide is the viral polypeptide, while in anotherembodiment, the N-terminal polypeptide is the viral polypeptide and theC-terminal polypeptide is the heterologous polypeptide.

ii. Proteases for Use with the Linkers

Some proteases useful according to the invention are discussed in thefollowing references: V. Y. H. Hook, Proteolytic and cellular mechanismsin prohormone and proprotein processing, RG Landes Company, Austin,Tex., USA (1998); N. M. Hooper et al., Biochem. J. 321: 265-279 (1997);Z. Werb, Cell 91: 439-442 (1997); T. G. Wolfsberg et al., J. Cell Biol.131: 275-278 (1995); K. Murakami and J. D. Etlinger, Biochem. Biophys.Res. Comm. 146: 1249-1259 (1987); T. Berg et al., Biochem. J. 307:313-326 (1995); M. J. Smyth and J. A. Trapani, Immunology Today 16:202-206 (1995); R. V. Talanian et al., J. Biol. Chem. 272: 9677-9682(1997); and N. A. Thornberry et al., J. Biol. Chem. 272: 17907-17911(1997). A variety of different intracellular proteases useful accordingto the invention and their recognition sequences are summarized inTable 1. TABLE 1 Properties Of Some Proteases Associated WithPost-Translational Processing Subcellular Tissue Protease LocalizationDistribution Cleavage Signal furin Golgi ubiquitous RXKR MMP-2 Golgitumor cells PLGLWA MT1-MMP plasma membrane tumor cells PLGLWA caspase-1secretory pathway ubiquitous YEVDGW caspase-2 secretory pathway VDVADGWcaspase-3 secretory pathway VDQMDGW caspase-4 secretory pathway LEVDGWcaspase-6 secretory pathway VQVDGW caspase-7 secretory pathway VDQVDGWalpha-secretase secretory pathway ubiquitous amyloid precursor protein(APP) proprotein convertase endoplasmic ubiquitous brain neurotrophicgrowth (subtilisin/kexin isozyme reticulum factor precursor (RGLT)SKI-1) proprotein convertases secretory pathway ubiquitous (PC-2, PC-3,etc.) tumor associated trypsin tumor cells foot and mouth diseaseNFDLLKLAGDVESNPGP virus, protease 2A

While not intending to limit the scope of the invention, the followingdescribes known proteases which might also be targeted by the linker,and their location in the cell. Proteases involved in the secretorypathway (ER/Golgi/secretory granules) includes, but are not limited to:Signal peptidase; Proprotein convertases of the subtilisin/kexin family(furin, PC1, PC2, PC4, PACE4, PC5, PC); Proprotein convertases cleavingat hydrophobic residues (e.g., Leu, Phe, Val, or Met); Proproteinconvertases cleaving at small amino acid residues such as Ala or Thr;Proopiomelanocortin converting enzyme (PCE); Chromaffin granule asparticprotease (CGAP); Prohormone thiol protease; Carboxypeptidases (e.g.,carboxypeptidase E/H, carboxypeptidase D and carboxypeptidase Z); and,Aminopeptidases (e.g., arginine aminopeptidase, lysine aminopeptidase,aminopeptidase B).

Cytoplasmic proteases include, but are not limited to: Prolylendopeptidase; Aminopeptidase N; Insulin degrading enzyme; Calpain; Highmolecular weight protease; and, Caspases 1, 2, 3, 4, 5, 6, 7, 8, and 9.For cytoplasmic proteins, it is necessary to use cleavage signals thatare recognized by cytoplasmic proteases and to use heterologous markerpeptides which have an appropriate hydrophilic/hydrophobic balance sothat they can escape across the plasma membrane. For marker peptidesthat must escape the cell via diffusion across the cell membrane, smallmolecular size (e.g., ≦10 kDa) will advantageously promote egress of thepeptide to the interstitial space.

Cell surface proteases or those occurring in the pericellular spaceinclude, but are not limited to: Aminopeptidase N; Puromycin sensitiveaminopeptidase; Angiotensin converting enzyme; Pyroglutamyl peptidaseII; Dipeptidyl peptidase IV; N-arginine dibasic convertase;Endopeptidase 24.15; Endopeptidase 24.16; Amyloid precursor proteinsecretases alpha, beta and gamma; Angiotensin converting enzymesecretase; TGF alpha secretase; TNF alpha secretase; FAS ligandsecretase; TNF receptor-I and -II secretases; CD30 secretase; KL1 andKL2 secretases; IL6 receptor secretase; CD43, CD44 secretase; CD16-I andCD16-II secretases; L-selectin secretase; Folate receptor secretase; MMP1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, and 15; Urokinase plasminogenactivator; Tissue plasminogen activator; Plasmin; Thrombin; BMP-1(procollagen C-peptidase); ADAM 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11;and, Granzymes A, B, C, D, E, F, G, and H.

iii. Self-Cleaving Linkers

An alternative to relying on cell-associated proteases is to use asequence encoding a self-cleaving linker. In one embodiment of theinvention, the foot and mouth disease virus (FMDV) 2A protease is usedas a linker. This is a short polypeptide of 17 amino acids that cleavesthe polyprotein of FMDV at the 2A/2B junction. The sequence of the FMDV2A propeptide is NFDLLKLAGDVESNPGP. Cleavage occurs at the C-terminus ofthe peptide at the final glycine-proline amino acid pair and isindependent of the presence of other FMDV sequences and cleaves even inthe presence of heterologous sequences.

Insertion of this sequence between two protein coding regions (i.e., thetwo halves of the fusion) results in the formation of a self-cleavingchimera which cleaves itself into a C-terminal fragment which carriesthe C-terminal proline of the 2A protease on its N-terminal end, and anN-terminal fragment that carries the rest of the 2A protease peptide onits C-terminus (see, e.g., P. deFelipe et al., Gene Therapy 6: 198-208(1999)). Thus, instead of using a cleavage signal recognizable by acell-associated protease, in this embodiment, the self-cleaving FMDV 2Aprotease sequence links the heterologous marker polypeptide to the viralpolypeptide resulting in spontaneous release of the marker polypeptidefrom the viral polypeptide.

iv. The Viral Polypeptide Portion of the Fusion

The measles virus offers 6 six target viral polypeptides from which tocreate a fusion protein, the N (nucleocapsid) protein, P proteins(polymerase cofactor phosphoprotein), M (matrix) proteins, F (fusion)proteins, H (hemaglutinin) protein and L (large; RNA polymerase)protein. The Rubulaviruses offer seven target viral polypeptides, whilethe pneumoviruses offer ten. Each virus (measles, rubulavirus, andpneumovirus) encode, in order, at least N, P, M, F, H. and L proteins.

Viral proteins which are particularly useful fusion partners for aheterologous marker peptide include the F and H proteins which areexpressed on the surface of the viral particle. When F and H proteinsare used, cleavable linkers are provide a way to introduce the markerpolypeptide into bodily fluids without reducing the bioactivity of the For H protein being used. This is especially important because thesyncytium-inducing abilities of Paramyxoviridae viruses reside in itsmembrane glycoproteins, which are responsible for mediating binding andfusion. Thus, the therapeutic or oncolytic activities of the virusreside in the F and H protein. In one embodiment of the invention, the For H proteins are used to create fusion proteins. Sequence informationrelating to F and H proteins from a variety of paramyxoviruses can befound in Morrison T. and Portner A. “Structure, function, andintracellular processing of the glycoproteins of Paramyxoviridae.” InThe Paramyxoviruses, Kingsbury, D. W. ed., Plenum Press, New York andLondon. (1991).

In one embodiment of the invention, a cleavable linker is providedcomprising a protease site for the same protease that activates the Fprotein, the subtilisin-like endoprotease furin in the trans-Golginetwork for cleavage activation of the F protein. In one embodiment ofthe invention, an H protein is fused to a marker heterologouspolypeptide through a linker comprising a furin cleavable site.Furin-cleavable marker polypeptides are therefore useful according tothe invention when fused to proteins, such as F and H proteins, that areprocessed through the Golgi network. Fusing to H protein has been doneby fusing the linker sequence to the extreme C-terminal residue of theH-protein. However, it is also possible to remove a few of theC-terminal residues (e.g., 0-20) and fuse onto the truncated C-terminus.

Although describing fusions between F and H proteins and heterologousmarker polypeptides, any Paramyxoviridae virus protein, may be used as afusion partner for a cleavable polypeptide as long as the fusion doesnot disrupt functions necessary for the replication of the virus in ahost cell. In this embodiment, the heterologous marker can be cleavedfrom its fusion partner either within the host cell or external to thecell following display on the cell surface.

3. Adding Marker Polypeptide Cistrons

An alternative to the expression of a marker polypeptide as a cleavablefusion protein is to link the marker polypeptide sequence to a viralprotein transcript through an internal ribosome entry site sequence(IRES). IRESs (also called ribosomal landing pads) are sequences thatenable a ribosome to attach to mRNA downstream from the 5′ cap regionand scan for a downstream AUG start codon, for example in polycistronicmRNA. See generally, Miles et al., U.S. Pat. No. 5,738,985 and N.Sonenberg and K. Meerovitch, Enzyme 44: 278-91 (1990). Addition of anIRES between the coding sequences for a viral gene product and themarker peptide can enable the independent translation of either theviral gene product or the marker peptide from a dicistronic orpolycistronic transcript.

IRES sequences can be obtained from a number of RNA viruses (e.g.,picornaviruses, hepatitis A, B, and C viruses, and influenza viruses)and DNA viruses (e.g., adenovirus). IRES sequences have also beenreported in mRNAs from eukaryotic cells (Macejak and Sarnow, Nature 353:90-94 (1991) and Jackson, Nature 353: 14015 (1991)).

Viral IRES sequences are detailed in the following publications: (a)Coxsackievirus: Jenkins, O., J. Gen. Virol. 68: 1835-1848 (1987);Iizuka, N. et al., Virology 156: 64-73 (1987); and Hughes et al., J.Gen. Virol. 70: 2943-2952 (1989); (b) Hepatitis A virus: Cohen, J. I. etal., Proc. Natl. Acad. Sci. USA 84: 2497-2501 (1987); and, Paul et al.,Virus Res. 8: 153-171 (1987); (c) Poliovirus: Racaniello and Baltimore,Proc. Natl. Acad. Sci. USA 78: 4887-4891 (1981); and Stanway, G. et al.,Proc. Natl. Acad. Sci. USA 81: 1539-1543 (1984); (d) Rhinovirus:Deuchler et al., Proc. Natl. Acad. Sci. USA 84: 2605-2609 (1984);Leckie, G., Ph.D. thesis, University of Reading, UK; and Skern, T. etal., Nucleic Acids Res. 13: 2111 (1985); (e) Bovine enterovirus: Earleet al., J. Gen. Virol. 69: 253-263 (1988); (f) Enterovirus type 70,Ryan, M. D. et al., J. Gen. Virol. 71: 2291-99 (1989); (g) Theiler'smurine encephalomyelitis virus: Ohara et al., Virology 164: 245 (1988);and, Peaver et al., Virology 161: 1507 (1988); (h) Encephalomyocarditisvirus: Palmenberg et al., Nucl. Acids Res. 12, 2969-2985 (1984); and Baeet al., Virology 170, 282-287 (1989); (i) Hepatitis C Virus: Inchauspeet al., Proc. Natl. Acad. Sci. USA 88: 10293 (1991); Okamoto et al.,Virology 188: 331-341 (1992); and Kato et al., Proc. Natl. Acad. Sci.USA 87: 9524-9528 (1990); and (j) Influenza virus, Fiers, W. et al.,Supramol. Struct. Cell Biochem. (Suppl 5), 357 (1981).

4. Independent Translation of a Heterologous Marker Polypeptide

Another alternative to the expression of a marker polypeptide as acleavable fusion protein is to insert the sequence coding for the markerpolypeptide between the coding sequences of the virus, allowingexpression of the marker as an independent translation product. Forexample, the sequence encoding the marker may be inserted between the Hand L protein coding sequences of the Paramyxoviridae virus genome, forexample the MV genome. In this embodiment, the stoichiometry of themarker expression would be fixed relative to the expression of viralgenes. However, it is well known that transcripts derived from thepromoter-proximal end of the MV genome are more abundant than thosederived from the promoter-distal end of the genome due to a gradient inthe expression of shorter versus longer transcripts from the viralpromoter. That is, the further away a given coding unit is from theviral promoter, the lower will be the number of transcripts containingthat unit. Therefore, while the relative amount of a marker translatedfrom a sequence inserted into the Paramyxoviridae virus genome betweenviral coding units will be constant with regard to the expression ofviral proteins, the absolute amount of the marker transcript, and hencethe marker polypeptide, will be influenced by its placement in thegenome relative to the viral promoter. The further the coding unit isfrom the viral promoter, the lower will be the expression of the marker.

5. Assays for Heterologous Marker Polypeptides

Once the viral construct has been introduced into the patient, therelease of the peptide marker can be monitored to determine whether andhow much viral replication is occurring. A sample of an appropriatebiological fluid or secretion is obtained from the patient and theconcentration of the marker polypeptide in the fluid or secretion isdetermined. Any biological fluid or secretion known to the art can beemployed, e.g., blood, urine, saliva, cerebrospinal fluid, mucous, orfeces, but the choice of sample is likely to be determined by the targetlocation of the construct within the body and the expected route ofrelease and excretion of the marker polypeptide. Samples of thebiological fluid can be obtained at any desired time interval followingadministration of the viral construct in order to monitor theeffectiveness of transfection, the regulation of infection and/or theprogress of therapy.

The presence of the marker polypeptide in the biological fluid samplecan be evaluated by any qualitative or quantitative method known in theart. Immunologic assays such as ELISA or radioimmunoassay are preferredbecause of their specificity, sensitivity, quantitative results, andsuitability for automation. Such assays are readily available in mostmedical facilities for a number of possible marker polypeptides such asinsulin C-peptide and beta-HCG. Chromatographic methods such as HPLC,optionally combined with mass spectrometry, can also be employed. Otheranalytic methods are possible, including the use of specific colorreagents, thin layer chromatography, electrophoresis, spectroscopy,nuclear magnetic resonance, and the like. While it is generallypreferred that the marker polypeptide itself be non-functional, i.e.,that it not possess any significant biological activity which mightinterfere with the patient's physiology or therapy, in one embodimentthe marker polypeptide possesses an enzyme activity is quantified andused as a means of detecting the marker in a biological fluid sample.

In embodiments where the marker polypeptide is a naturally occurringpeptide, such as a cleavage fragment of a peptide hormone precursor, thebackground level of the peptide is determined prior to administration ofthe construct and is simply subtracted from the value determined afterinfection to provide a measure of marker polypeptide released throughexpression from the therapeutic virus. In the embodiment where themarker polypeptide is naturally present in the patient and fluctuateswith physiological or pathological circumstances, the background rhythmor cycle of the marker is determined to estimate and subtract fromvalues determined post infection. However, in another embodiment, thebackground level of the marker polypeptide or its fluctuations issuppressed through drugs, modification of the patient's diet, or othersuitable measures.

Depending on the degree of accuracy required, the level of expression ofthe virus can either be inferred from the concentration of markerpolypeptide determined in a biological fluid sample or can be determinedmore accurately by calibration. The level of expression of a virusprotein is expressed as the amount of such protein, in moles or mg,synthesized by the cell, tissue, organ, or entire organism which was thetarget of the viral therapy per unit time. For example, the level ofexpression can be quantified as the number of nanomoles of a particularviral protein produced per gram of tissue per hour.

In one embodiment, calibration of the marker polypeptide is performed byquantifying both the marker polypeptide and a viral protein productitself (e.g., by extracting the tissue making the viral product andmeasuring the product directly using HPLC, ELISA, radioimmunoassay,Western blot, or other suitable method) over a sufficient time period topermit extrapolation or determination of the stoichiometry betweenmeasured marker polypeptide in a given biological fluid sample andactual tissue level of transgene product. Without calibration, astoichiometry must be estimated or assumed in order to accuratelydetermine expression of viral product. Even if an assumed stoichiometryis not 100% accurate, it should allow at least qualitative orsemi-quantitative tracking of viral activity.

B. Modifications to Enhance Cytotoxic Efficacy of a Paramyxovirus

The invention provides recombinant Paramyxoviridae viruses which have anenhanced ability to kill tumor cells. In one embodiment, aParamyxoviridae virus is provided which has enhanced fusogenicity. Inanother embodiment of the invention, a virus is provided which deliversa cytokine to a tumor cell, creating an enhanced immune response againstthe tumor cell in addition to the direct cytotoxic effect of the virus.

i. Enhanced Fusogenicity

In the course of a natural Paramyxoviridae virus infection, viral spreadand virus-mediated tissue damage proceed unchecked until the host immunesystem terminates the infection. Thus, one of the principles of cancerviral therapy is that there is a window of opportunity from the time ofvirus inoculation until the establishment of an effective immuneresponse during which the extent of tumor destruction will be dependenton the speed of virus propagation and the intrinsic cytotoxic potentialof the virus. Since the cytotoxic properties of Paramyxoviridae virusesreside largely in the ability of their surface glycoproteins to triggercell-cell fusion and hence syncytium formation, the enhanced potency ofinduction of cell-cell fusion by a specified Paramyxoviridae virusshould create an enhanced therapeutic effect.

The F (fusion) and H (hemagglutinin) proteins of Paramyxoviridae areresponsible for triggering cell-cell fusion. In cultured cell lines,co-expression of paired membrane glycoproteins is required for syncytiuminduction, although some exceptions have been observed, such as SV5,whose F protein alone appears sufficient for syncytium induction.Paramyxoviridae virus F proteins are initially synthesized aspolyprotein precursors F₀ which cannot be activated to trigger membranefusion until they have been proteolytically cleaved, usually by a serineprotease in the Golgi compartment. The protease cleaves the F₀ precursorto yield an extraviral F₁ domain and a membrane anchored F₂ domain,which remain covalently associated through disulphide linkage.Activation of this processed form of F to trigger membrane fusion isbelieved to result from binding of the attachment protein(hemagglutinin, H, for measles virus) to the cellular receptor, aninteraction which induces conformational changes in H and in turn in F,thus exposing on it a hydrophobic fusion peptide which inserts into thecellular membrane, thereby initiating fusion.

The activity of F and H proteins has been shown to be regulated by the M(matrix) protein which interacts with their cytoplasmic tails. Measlesviruses in which the interaction between the M and F&H glycoproteins hasbeen disrupted, either by deletion of M or by the deletion of thecytoplasmic tails of F&H, have been shown to induce more potentcell-cell fusion (Cathomen 1998 EMBO 17 3899, Cathomen 1998 JV 72 1224).

Similarly, truncation of the cytoplasmic domains of a number ofretrovirus and herpesvirus glycoproteins has been shown to increasetheir fusion activity, sometimes with a simultaneous reduction in theefficiency with which they are incorporated into virions (Rein et al.,1994, J. Virol. 68: 1773; Brody et al., 1994 J. Virol. 68: 4620;Mulligan et al., 1992, J. Virol. 66: 3971; Pique et al., 1993, J. Virol.67: 557, Baghian et al., 1993, J. Virol. 67: 2396; Gage et al., 1993, J.Virol. 67: 2191). Viruses of the Paramyxoviridae family withdestabilised matrix/envelope interactions display a marked reduction intheir release from infected cells (Cathomen et al., 1998, EMBO J. 17:3899), presumably reflecting a reduced ability to form orthodox viralparticles. This modification thus imparts two distinct advantages forcancer therapy: firstly, increased local spread and hence cell killing,and secondly, reduced release concomitant with reduced systemic viralspread.

Thus, in one embodiment of the invention, Paramyxoviridae variants withmodified fusogenicity are generated for use in cancer therapy bymodification of any one, two, or all three of the H, F and M proteins;however, it is necessary to co-express H protein with F protein forParamyxoviridae virus cell fusion activity.

a. Modified H Proteins

The H protein cytoplasmic tail comprises the amino-terminal 34 aminoacids of the protein (sequence: NH₂—MSPQRDRINAFYKDNPHPKGSRIVINREHLMIDR-COOH). Modification of H protein byremoval of the 24 amino acids immediately following the initiatormethionine (AA 2-25) results in a loss of fusogenic activity by thevirus. In contrast, deletion of either 8 amino acids immediatelyfollowing the initiator methionine (amino acids 2-9 deleted) or 14 aminoacids between amino acids 2 and 17 (amino acids 3-16 deleted) enhancethe fusogenic activity of the virus (Cathomen et al., 1998, J. Virol.72: 1224). These results indicate that membrane proximal amino acids 17to 25 comprise a sequence necessary for fusion activity. The resultsalso indicate that amino acids at least between those numbered 2-16 areinvolved in negative regulation of fusogenic activity. Therefore, in oneembodiment, fusogenicity is enhanced by deletion of either amino acids2-9, or 3-16. In a further embodiment of the invention, amino acids 2-16are deleted. In still a further embodiment of the invention, amino acids2-24 are deleted, and preferably 2-20. In one embodiment of theinvention, the virus comprises a truncated H sequence comprising 8-14fewer amino acids.

In a further embodiment of the invention, any or all of amino acids 2-16are modified by systematically deleting and/or substituting amino acidsand assaying for mutations which increase the fusogenicity of the virus(as measured by determining an increase in the number of cells havinggreater than 20 nuclei per cell after infection at a given multiplicityof infection with a modified or unmodified virus). In one embodiment ofthe invention, substitutions are selected which do not disrupt the size,charge, and/or hydrophobic character of the H protein relative to thewild-type sequence.

b. Modified F Proteins

In one embodiment, the F protein is modified by alteration of itscytoplasmic tail, which comprises the carboxy-terminal 33 amino acids ofthe protein (sequence: NH₂— RGRCNKKGEQVGMSRPGLKPDLTGTSKSYVRSL-COOH).Modifications to the F protein found to increase the fusogenic activityof the virus include addition of unrelated sequences to the C-terminus(for example, by alteration of the normal stop codon, or other means),and deletion of 16 or 24 C-terminal amino acids (Cathomen et al., 1998,J. Virol., supra). Viruses incorporating these changes induce fasterformation of syncytia, with a concomitant enhancement in the rate ofcell killing. In another embodiment of the invention, a modified virusis provided which comprises a C-terminal tail comprising at least onedeletion and/or substitution and which has enhanced fusogenic activityand/or cell killing effects. The addition or exchange of sequences havealso been demonstrated to enhance the fusogenicity of the virus.Therefore, in a further embodiment of the invention, the F protein ismodified by the addition of 28 amino acids, while in still a furtherembodiment, the C-terminal tail of the F protein is exchanged with theC-terminal tail of a Sendai virus.

In another embodiment of the invention, the virus comprises mutations inboth the F and H protein. Viruses which have been generated comprisingan H protein mutant (animo acids 3-16 deleted) and an F proteincomprising either 28 amino acids of extraneous sequence, a deletion of24 C-terminal amino acids or a replacement of the tail with acytoplasmic tail derived from Sendai virus all exhibited enhancedfusogenic activity relative to wild-type virus.

C. Modified M Proteins

In another embodiment of the invention, a Paramyxoviridae family virusis provided having an altered or deleted M protein to obtain a virushaving enhanced fusogenicity. The alteration of the M protein accordingto this aspect of the invention may be either wholesale deletion of theprotein, or alternatively, one may delete or substitute amino acidsnecessary for the association of F and H glycoproteins. This may beaccomplished by techniques known in the art for systematic site-directedmutagenesis. Clones of Paramyxoviridae virus bearing mutations in the Mprotein are monitored for enhanced fusogenic activity according tomethods known in the art or described herein below.

In order to assess the effect of a modification to the F, H and/or Mproteins on the fusogenic activity of the virus, cells are infected inculture and monitored for the formation of syncytia over time relativeto syncytia formation occurring with wild-type virus or a referencevirus lacking modified F, H and/or M proteins. In another embodiment,mice are infected intracerebrally (see, e.g., Cathomen et al., EMBO J.17: 3899, 1988) and monitored for viral penetration by in situhybridization or immunohistochemistry to detect viral RNA or proteins.

d. Assays for Fusogenic Activity

Fusogenicity is said to be increased or enhanced if the number ofnuclei/per syncytium is greater by about 10%, preferably by 20%, 35%,50%, 100%, 200% up to 500% or more than the number of nuclei persyncytium observed at a given time after infection with an unmodifiedvirus of the same strain as the modified virus when the infection isperformed at the same multiplicity of infection. Measurement of thenumber of nuclei in a syncytium or the number of syncytia is most easilyaccomplished via in vitro (or ex vivo) assays, as described herein.

A culture assay for fusogenic activity may be performed as follows byisolating infectious virus particles by rescue from transfected cellsexpressing helper functions. One method known in the art for rescuinginfectious MV variants from transfected cells expressing helperfunctions was described by Radecke et al. (1995, EMBO J. 14: 5773,incorporated herein by reference). In this method, recombinant virusesare generated by co-transfecting a recombinant viral (RNA) genome,encoded on a plasmid under the direction of a T7 phage promoter, with aplasmid encoding full length L protein into a human embryonic kidneycell line (293 cells) stably expressing T7 RNA polymerase and MV N and Pproteins (the construction of such a helper cell line (293-3-46) isdescribed in detail by Radecke et al., 1995, supra). Transcription ofthe plasmid-borne viral genome by T7 polymerase produces viral genomicRNA that is encapsidated by the viral proteins stably (N and P) ortransiently (L) expressed in the transfected helper cells. The L proteinis expressed transiently, rather than stably since high levels of Lexpression can impair the rescue of virus; transient expression allowstitration of the L protein as needed.

The Paramyxoviridae virus genomic plasmid has the followingcharacteristics. The T7 promoter allows the production ofParamyxoviridae virus antigenomic RNA starting precisely with the firstParamyxoviridae virus nucleotide. The hepatitis delta virus ribozymefollows the Paramyxoviridae virus genomic sequences such that theribozyme cleaves the RNA at the Paramyxoviridae virus 3′ terminus.Following the ribozyme sequence is a T7 polymerase termination sequenceto ensure that adjacent vector sequences do not interfere with ribozymecleavage activity. One important factor for the proper assembly andfunction of recombinant viruses is that the total number of nucleotidesin the genomic replicons should be a multiple of six, to adhere to theso-called “rule of six”, originally identified in Sendai virusreplication (Calain and Roux, 1993, J. Virol. 67: 4822). Theconstruction of the p(+)Paramyxoviridae virus plasmid is described indetail by Radecke et al. (1995, supra), as is the generation of the P, Nand L plasmids that supply the helper functions.

For the rescue of recombinant viruses, 293-3-6 cells are transfected in35 mm dishes with 8 ug of a plasmid encoding a modified Paramyxoviridaevirus genome (derived, for example, from the Paramyxoviridae virusgenomic plasmid p(+)Paramyxoviridae virus) in the presence of 5 ng ofplasmid encoding the Paramyxoviridae virus L polymerase (for example,pEMC-La; Radecke et al., 1995, supra). Two days after transfection,cells are expanded from 35 mm to 90 mm dishes and cultured another twodays before scraping and adsorption to Vero cell monolayers. InfectedVero cells are monitored for syncytia formation, and syncytia are pickedand propagated further on Vero cell cultures. Virus is harvested fromVero cell syncytia by scraping cells from the dishes and subjecting themto two rounds of freeze/thaw. The cleared supernatants represent “plaquepurified” virus. Viral stocks are produced by infection of Vero cellmonolayers (adsorption for 1.5 hours at 37° C.). followed by scraping ofinfected cells into the medium and freeze/thaw lysis. Viral stocks arealiquotted, frozen and stored at −80° C.

In another embodiment, a second, novel method of rescuing infectiousviral variants is provided. In this embodiment, susceptible cells areinfected and monitored for the formation of syncytia. Modified virusesisolated as in step (a) above are assayed for enhanced fusogenicity byinfection of cultured African green monkey kidney (Vero) cells, whichgrow on Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5%fetal calf serum (FCS). Recombinant viral titers are assayed for byinfecting Vero cells in 35 mm culture dishes. After 2-3 hours of viraladsorption, the inoculum is removed and cells are overlaid with 2 mlDMEM containing 5% FCS and 1% SeaPlaque agarose. After 3 to 5 days,cultures are fixed with 1 ml of 10% trifluoroacetic acid for 1 hour,then UV cross-linked for 30 minutes. After removal of the agaroseoverlay, cell monolayers are stained with crystal violet and plaques arecounted to determine viral titer.

In one embodiment, enhanced fusogenicity of recombinant viruses isassayed for by infection of separate cultures of Vero cells with eitherthe recombinant or the wild-type virus (or any modified viral referencestrain used as the starting point in generating a particularrecombinant) at the same multiplicity of infection (MOI). Cell fusionactivity of the virus is evaluated by monitoring the number and size ofsyncytia (number of nuclei per syncytium) over time. Similarly,cytotoxic activity of the viruses can be monitored by following thedeath of cells over time. A particular recombinant is said to exhibitenhanced fusogenicity according to the invention if the engineered virusyields greater than about 125%, preferably 135%, 150%, 200%, or up to ashigh as 500% or more of the number of nuclei per syncytium observed withwild-type or reference virus at a given time after infection relative tothat observed with the appropriate control virus. Similarly, thecytotoxicity of a particular recombinant or engineered virus is said tobe enhanced if the rate of cell death is about 125%, preferably 135%,150%, 200%, or up to as high as 500% or more of the rate observed withwild-type or reference virus at a given time after infection relative tothat observed with the appropriate control virus.

ii. Viral Expression of Immunomodulatory Proteins.

In another embodiment of the invention, host antitumor activity isstimulated by providing a virus capable of expresssing animmunostimulatory virus (e.g., a cytokine). Because it has beendemonstrated that exogenous genes may be introduced to theParamyxoviridae virus genome (e.g., by insertion between viral proteincoding sequences or by expression as protease-cleavable fusion proteins,as described above), Paramyxoviridae virus variants expressing exogenousproteins that potentiate the killing of tumor cells are useful toenhance the efficacy of anti-tumor activity. Proteins able to potentiatethe killing of tumor cells include those cytokines or otherimmunostimulatory proteins that stimulate a cell-mediated anti-tumorimmune response by recruiting immune cells to the site of cytokineproduction.

Cytokines or immunostimulatory proteins useful according to this aspectof the invention include, but are not limited to, the following (thenumber following each cytokine is the GenBank Accession No. for thesequence encoding the cytokine): IL-1, M28983; IL-2, S77834; IL-3,M14743; IL-4, M13982; IL-5, J03478; IL-6, M54894; IL-7, J04156; IL-12,AF101062; IFN-γ, U10360; and TNF-, M16441 and any other protein thatstimulates the immune response (e.g., a costimulatory molecule).

The expression of immunomodulatory protein by a modified Paramyxoviridaevirus according to the invention may be assessed in infected cellcultures by means known in the art for assaying the presence of theparticular protein. For example, expression of immunomodulatory proteinmay be evaluated by Western (immunoblot) analysis using antibodiesrecognizing the specific protein. Other immunoassays, such as ELISAs maybe used, or, alternatively, cell-based assays for the activity of theprotein may be used as known in the art.

An enhanced immune response is assessed by assays for, e.g., antibodyproduction, lymphocyte proliferation, cell-mediated cytotoxicity, orcytokine production which is significantly higher in an organism treatedwith the modified virus when compared to an organism treated with theunmodified virus (to within 95% confidence levels) and/or has aa reducedincidence of tumor formation (e.g., from 100% to 10%) in a mammalreceiving the modified virus.

C. Modifications that Enhance the Selectivity of Viral Infection

Improvements in the selectivity of Paramyxoviridae virus infectionenhance its usefulness as a vector for oncolytic therapy since suchimprovements minimize damage to surrounding, non-tumor tissue. In oneembodiment, to prevent collateral damage to normal host tissues,modifications that serve to limit virus spread and viral cytotoxicity tothe microenvironment of the neoplastic cells may be introduced to theParamyxoviridae virus genome. In one embodiment, the infective activityof the virus is made dependent upon an activity, such as a protease,associated with the tumor microenvironment. In a second embodiment, thevirus is targeted to a tumor-specific protein.

1. Restricting Paramyxoviridae Viral Infectivity by Making it Dependenton Activities Associated with the Tumor Microenvironment

The F glycoprotein of Paramyxoviridae viruses is critical for triggeringof both virus-cell and cell-cell fusion. As discussed above, the proteinis synthesized as a precursor, F₀, which is proteolytically cleaved intoF₁ and F₂ components by a ubiquitous Golgi compartment protease (furin).This cleavage is necessary for activation of the fusion function of theprotein, and hence for the infectivity of the virus.

In one embodiment, the infectivity of the virus is restricted largely totumor cells by making its proteolytic activation dependent on atumor-associated protease. Proteases such as matrix-metalloproteinases(MMPs), plasminogen activator/plasmin system, p65, cathepsins,trypsin-like proteases, human kallikrein 2 and prostate specific antigenare intimately involved in cancer invasion and metastasis and incomplement resistance, and tumors therefore provide a protease-richmicroenvironment.

The invention thus contemplates the introduction of selectedcancer-associated protease cleavage sites into viruses. The cleavage ofthe F₀ precursor may be made dependent on a protease other than furin byreplacement of the furin cleavage signal R—R—H—K—R at amino acids 108 to112 of the measles virus (MV) or the corresponding residues of otherParamyxoviridae viruses with that of another protease. Cleavage by furinoccurs after arginine 112. Correct cleavage at this site is essential,because changing arginine 112 to leucine has been shown to result inaberrant cleavage and loss of fusion ability (Alkathib et al., 1994, J.Virol. 68: 6770).

Proteases with sites useful for restricting the infectivity of atherapeutic virus according to the invention include, but are notlimited to those listed in Table I. The cloning of the Paramyxoviridaevirus F protein into an expression vector, pCG, under control of the CMVearly promoter to generate the plasmid pCG-F was described by Cathomenet al. (1995, Virology 214: 628). Site-directed mutagenesis to convertthe furin cleavage site to a site for another protease may beaccomplished by one of skill in the art using any of a number ofsite-directed mutagenesis methods known in the art. One example of asite-directed mutagenesis approach is that embodied by the QuikChangeSite-Directed Mutagenesis Kit (Stratagene, La Jolla, USA), which usestwo complementary mutagenic primers and a double-stranded plasmidtemplate.

Paramyxoviridae viruses modified to restrict infectivity by changing theF₀ protease cleavage site may be assessed for restricted infectivity byinfecting cultured cells in the presence and absence of the protease, ifit is an extracellular protease. For an intracellular proteaseassociated with a given tumor cell type, cells either expressing or notexpressing the protease are infected with recombinant virus. In oneembodiment, the infectivity of the virus is considered to be modified ifthe virus infects (i.e., causes the formation of syncytia or plaques,monitored as described for viral titer assays) cells either in thepresence of or expressing the specific protease more efficiently than itinfects cells either not expressing or in the absence of the specificprotease. As used in this context, “more efficiently” or “to a greaterdegree” refers to a number of syncytia or plaques formed in a givenamount of time that is at least 1.25-fold, and preferably 10-fold,100-fold, 1000-fold or greater in the cells expressing or cultured inthe presence of the protease, relative to cells not expressing or notcultured in the presence of the protease.

Another means of evaluating modified viruses for restriction of hostspecificity is to perform infections of cultured cells expressing theprotease activity used to re-target the specificity in the presence andabsence of inhibitor(s) of the targeting protease. A recombinant virusthat only infects or that infects to a greater degree in the absence ofthe inhibitor may be said to be restricted in its host specificityaccording to the invention. The most efficiently that a virus may betargeted is the situation where it is completely dependent upon thepresence of the targeting protease for infection and does not infect atall in the absence of the protease.

The invention also contemplates generating Paramyxoviridae virusesexpressing F proteins activatable by cancer-specific proteases throughthe production of a randomized cleavage signal library and selection offunctional protease-activatable viruses bearing F protein mutations in avariety of tumor cells. In order to generate a randomized cleavagelibrary, PCR primers (an F primer and a random sequence primer) are usedto amplify an F₀ plasmid template.

The F primer should have sequences complementary to the F₀ templatesequence 3′ of the nucleotides encoding the furin cleavage sequence(i.e., complementary to F₁ sequence adjacent to the cleavage site),preceded by (i.e., 3′ of) a randomized stretch of 12 bases correspondingin position to those encoding the furin cleavage site RHKR. In order toavoid restoring furin cleavage, the “randomized” stretch of nucleotidesshould not be truly random, but rather should be designed such thatpositions 1, 2 and 3 cannot be lysine or arginine (see Example 5).

The “random” sequence primer and the downstream sequence PCR primershould also incorporate restriction sites allowing the sub-cloning ofthe fragments generated into a retroviral transfer vector. Followingamplification using the F₀ template, the library of PCR products isdigested with the appropriate enzyme(s) and cloned into the retroviraltransfer vector (for example pMFGnlsLacZ, wherein the nlsLacZ sequencesare removed by BamHI digestion) to generate a library of F proteincleavage site retroviral expression constructs. The library istransfected into a panel of tumor cell lines. By co-transfection of an Hexpressing plasmid, such as pCG-H (Cathomen et al., 1998, J. Virol.,supra), cells in which F is properly cleaved to expose theamino-terminal amino acids of F₁ are identified by their ability to formsyncytia, which can then be picked to isolate the particular cleavagesite mutant activatable by a protease expressed by that tumor cell line.

In one embodiment, the paramyxoviral library is used to infect a panelof human cell lines which are subsequently observed for the formation ofmultinucleated syncytia, expected to be maximal 24 to 72 hours afterinfection of the cells. The cell lines are grown to near-confluencybefore infection. Examples of cell lines that can be used for thisassay, include but are not limited to, A431 (epidermoid carcinoma),HT1080 (fibrosarcoma), EJ (bladder carcinoma), C175 (colon carcinoma),MCF7 (breast carcinoma), HeLa (cervical carcinoma), K422 (follicularlymphoma), U266 (myeloma).

DNA is extracted from each syncytium formed in a tumor cell line,followed by PCR amplification of the F sequence and sequencing toidentify the cleavage signal sequence. Further selective pressure may beapplied to the system by transfecting the cells in the presence ofparticular protease inhibitors. By the nature of their selectionprocess, clones isolated from the cleavage library will be known to havemodified selectivity of host cell type.

2. Restricting Paramyxoviral Infectivity by Targeting to a SpecificCell-Surface Protein.

An alternative strategy to limit collateral damage to normal tissues isto engineer the binding specificity of the Paramyxoviridae virusmembrane glycoproteins to restrict the specificity of virus-cell andcell-cell fusion. In such a case, virus propagation and virus-mediatedcytotoxicity will be confined to neoplastic tissues expressing thetargeted cell surface marker. Any tumor-specific cell-surface protein isuseful according to this targeting method, although it is preferred thatthe protein not be sequestered from potentially binding ligands. It hasbeen demonstrated that polypeptide binding domains can be displayed onthe surface of Paramyxoviridae viruses or Paramyxoviridae virus-infectedcells as C terminal extensions of the H glycoprotein.

In one embodiment the displayed ligand positively retargets the virus tocancerous cells via the specific targeted cell surface marker, mediatingboth viral attachment and entry. Paramyxoviridae family viruses aremodified using bispecific antibodies which prevent attachment to theviral receptor and instead confer new binding specificities, resultingin target cell transduction. Successful attachment and infection hasbeen demonstrated for adenoviruses targeted to EGF (Watkins et al.,1997, Gene Ther. 4: 1004), folate receptor (Douglas et al., 1996 NatureBiotech. 14: 1574), and FGF-2 (Goldman et al., 1997, Cancer Res. 57:1447).

Alternatively, the invention contemplates Paramyxoviridae virusesengineered such that targeted attachment of the virus to the cell doesnot lead to gene delivery, unless protease cleavage occurs.

For Paramyxoviridae viruses, the choice of targeted receptor plays arole in determining the success of gene transfer. A receptor whichsequesters the virus away from the cell surface will not permit fusion,precluding gene delivery. Attempts to extend the host range of ecotropicMLV through the display of polypeptides such as stem cell factor (SCF,Yajima et al., 1998, Hum. Gene Ther. 9: 779, Fielding et al., 1998,Blood 91: 1802), human MHC-I (Marin et al., 1996, J. Virol. 70: 2957),erythropoetin (Kasahara et al., 1994, Science 266: 1373), EGF (Cossettet al., 1995, J. Virol. 69: 6314), anti-CD3 antibody (Ager et al., 1996,Hum. Gene Ther. 7: 2157), anti-colon carcinoma antibody (Ager et al.,1996, Hum. Gene Ther. 7: 2157) and anti-human LDLR single chain antibody(Somia et al., 1995, Proc. Natl. Acad. Sci. USA 92: 7570) on or as partof the envelope glycoprotein have resulted in recombinant viruses whichcan bind the targeted receptors but which give very low levels oftransduction in the target cells. For Paramyxoviridae viruses, directentry at the cell surface occurs, and this is believed to be triggeredby receptor binding which induces conformational changes in the envelopeglycoproteins such that a hydrophobic domain on the fusion protein isexposed.

The present invention circumvents this limitation, through the use ofprotease activatable viruses. In one embodiment, a (preferablytumor-specific) protease cleavage site is placed between the envelopeglycoprotein and the displayed ligand, such that subsequent to targetedattachment, cleavage of the displayed ligand fully exposes the envelopeglycoprotein and allows virus-cell membrane fusion to proceed. In thisstrategy, the degree of selectivity may be enhanced by the dependence ontwo tumor-specific targets: the cell surface ligand and the protease(see Table I for a non-limiting list of proteases useful for targeting).

In one embodiment, ligands that bind tumor-specific cell surfaceproteins are expressed as cleavable fusions with Paramyxoviridae virus Hproteins using the same approach as used to generate Paramyxoviridaevirus H protein tagged with a marker peptide described above (also seeExample 1). For example, an SfiI site in the MV H protein or anotherrestriction site in about the same region of the gene can be engineeredby means well known in the art to create a site for introduction of thesequence of a cleavable ligand. One of skill in the art may modify theSfiI site as necessary to accommodate a given ligand coding sequence.Any ligand known to bind a tumor specific antigen may be fused to Hprotein and displayed on the surface of the modified virus. In oneembodiment, the ligand is fused to H protein by a linker that issensitive to a tumor-associated protease. Ligands for tumorcell-specific surface proteins include, but are not limited to,single-chain antibodies that recognize a given tumor antigen. Sequencesencoding a single chain antibody can be introduced as fusions with the Hprotein in the same manner as sequences for other ligands.

In one embodiment, the effect of the display of a targeting ligand as anH fusion on the selectivity of a modified Paramyxoviridae virus isassessed by infection of cells either expressing or not expressing thetumor-specific protein bound by the ligand. A modified virus is said tohave increased specificity or selectivity if it infects cells expressingthe specifically targeted tumor protein more efficiently than it infectscells lacking the specifically targeted tumor protein. The most specifica targeted infection may be is when the virus is completely dependent onthe presence of the tumor-specific protein on the cell surface forinfection.

D). Modifications of Paramyxoviridae Viruses that ReduceTransmissibility of the Virus for Oncolytic Viral Therapy

For safety reasons, it is desirable that an engineered Paramyxoviridaevirus used for oncolytic viral therapy should not be transmitted fromthe treated patient to care givers, relatives or sexual partners. Tothis end, engineered vaccine strains of Paramyxoviridae viruses whichare known to have low transmissibility compared to their more highlypathogenic counterparts are suitable for use according to the invention.In addition, the previously described Paramyxoviridae virusmodifications that disrupt the interaction between M and F and H andenhance the potency of cell-cell and virus cell fusion, are associatedwith a reduction in the efficiency with which virus is released frominfected cells. Hence, modifications of this kind appear to enhancelocal spread of virus between neighboring cells while simultaneouslyreducing the risk of systemic spread and transmissibility between hosts.Naturally existing strains of virus which have low transmissibility arealso encompassed within the scope of the invention, and encompass theEdmonston strain and the Moraten strain of measles virus.

E. Production of Genetically Altered Paramyxoviridae Viruses

A convenient and reliable method for the generation of Paramyxoviridaevirus recombinants for oncolytic viral therapy is disclosed to introduceany, some, or all of the modifications described above. In this improvedmethod, the desired modifications (e.g., those providing markerexpression, enhanced syncytium formation, enhanced cell-mediatedimmunostimulation, enhanced selectivity of infection and/or reducedtransmissibility) are engineered into a plasmid comprising an infectiousmolecular clone of the Paramyxoviridae virus genome sequence. Expressionof the antigenomic viral RNA is under the direction of the T7 RNApolymerase promoter. A helper virus is then used to provide all of thecomplementing functions required to rescue the infectious clone intoviable Paramyxoviridae virus particles. Elimination of the helper virusgenome is facilitated by its unique characteristics. Namely, that thehelper virus may be deleted for, or expresses no F protein capable ofcooperating with H protein to promote virus-cell or cell-cell fusion.Alternatively, the helper virus may carry a trypsin activatable Fglycoprotein or it may carry sequences encoding a tag, such as GFP, foridentification. Repeated passage of recombinant virus in the appropriateselection system, i.e in the absence of trypsin or by selectingnon-fluorescing syncytia, respectively, results in the isolation ofvirions containing only the rescued genome with no contamination fromhelper virus genomes.

Helper viruses necessary for this approach provide N, P and L proteinsfor the replication and encapsidation of the modified virus intoinfectious particles. The helper virus must also be either deleted in F(and thus previously produced on F expressing cells), express atrypsin-activatable F (as described herein), or encode GFP (greenfluorescent protein) expression. Following transfection of the modifiedgenomic Paramyxoviridae virus plasmid into cells expressing T7polymerase (e.g., 293 cells stably transfected with a T7 polymeraseencoding plasmid; construction of a T7-Neo plasmid, pSC6-T7-NEO isdescribed by Radecke et al., 1995, supra), cells are infected with thehelper virus. N and P proteins coat the antigenomic, T7-transcribed RNA,which is then replicated by the viral polymerase L to produce a genomicRNA which is the template for subsequent mRNA transcription andproduction of viral proteins. The plasmid encoding the antigenomic RNAmust also encode a ribozyme, such as the hepatitis delta ribozyme,situated at the end of the transcript such that the proper ends of thevirus are generated.

Helper virus encoding a GFP tag is constructed as follows. The PlasmidpMeGFPNV includes the antigenome of MV with an insertion containing theopen reading frame of enhanced green fluorescent protein (eGFP) flankedby the 3′ and 5′ untranslated regions of the N gene. The cloningstrategies used in the generation of pMeGFPNV are as follows.

1. P(+)MPCATV#32 (see FIG. 2) is cleaved with Nru 1 in a one folddigestion. After sodium acetate/EtOH precipitation, the linearizedplasmid is digested with Mlu 1 and dephosphorylated. The plasmidbackbone is then separated form the excised CAT insert by agaroseelectrophoresis. pBLoT(+)GFP14 is digested in the same way asp(+)MPCATV#32. The excised GFP fragment was then isolated by agarose gelelectrophoresis and ligated with the backbone of p(+)MPCATV#32. Positiveclones are identified by Hpa 1 digestion of miniprep DNA and growth toobtain maxiprep DNA. Correct insertion of the GFP fragment is thenconfirmed by restriction analysis and sequencing. The resulting plasmidis designated as p(+)MPGFPV11.

2. The plasmid p(+)MPGFPV11 is digested with Nru 1, precipitated andcleaved with Mlu 1. After dephosphorylation of the 5′ ends, the backboneDNA is isolated by extraction from an agarose gel. The lrGFP cassette isexcised from the plasmid pBLoT(+)lrGFP6 by digestion with Mlu 1 and EcoRV. After isolation from an agarose gel, it is ligated to the previouslydigested p(+)MPGFPV11 backbone. The minipreps of this ligation are thenscreened by digestion with HpaI, and two clones, designed asp(+)MPlrGFPV1 and 7 (FIG. 3) are grown for DNA maxipreparation.Sequencing over the cloning sites is done to confirm the resultsobtained by the analytical digestions.

The Sfi I×Sac II cassettes of the plasmids p(+)MV-2A#12 (by F. Radecke)and p(+) MplrGFPV7 (see above) are exchanged for the same fragmentexcised from p(+)MlrGFPNP 3. For this purpose, all three plasmids arecompletely digested with both enzymes. In addition, the 5′ ends of thefull-length plasmids are desphosphorylated before all fragments ofinterest are purified by agarose gel electrophoresis. After ligation andtransformation, minipreps are grown and screened for positives bydigestion with Hpa 1.

Depending on the backbone used, the newly generated plasmids carry oneor two additional trancription units (ATUs) designed as p(+)MlrGFPNV14and 23 (NATU in front of the N gene) and p(+)M2xlrGFPV 38 and 45 (N- andP-ATU), respectively (FIG. 4).

The additional transcription unit (ATU) encoding GFP is comprised of 852nucleotides, and pMeGFPNV conforms to the rule of six. The insertion wasmade into the full length infectious clone of measles virus, p(+)MV(EMBL Accession No. Z66517) between the 3′ end and the gene encoding thenucleocapsid protein. Recombinant virus is recovered from this plasmidusing the 293-3-46 rescue cell line (Radecke et al., 1995, supra). Thecell line is transfected with pMeGFPNV (5 ug) and pEMCLa (10 ng), whichexpresses the MV polymerase protein under the control of the T7promoter, using a calcium phosphate transfection procedure. Cellmonolayers are monitored microscopically for the appearance of syncytiaeach day. Autofluorescence with these syncytia, indicating eGFPexpression, is verified using an inverted UV microscope (Leica). Virusstocks are produced following plaque-purification and titers ofapproximately 5×10⁵ TCID₅₀/ml are obtained.

Viruses produced from the 293-T7 cells are heterogeneous, containingeither rescued or helper virus genomes, or both. To separate rescuedvirus from helper in the case of the trypsin-activatable helper virus,the virus is serially passaged in the absence of trypsin. After about 10sequential passages in the absence of trypsin, virus will generally befree of helper.

In order to separate rescued virus from helper in the case of the Fdeleted helper virus, transfected 293-T7 cells are overlaid onto Verocell monolayers, syncytia are picked and the process repeated forseveral rounds of infection.

In order to separate rescued virus from helper in the case of theGFP-tagged helper virus, transfected 293-T7 cells are overlaid onto Veromonolayers and syncytia that do not fluoresce are picked and re-passagedon fresh cells. Repeated passage of non-GFP-expressing viruses selectsfor those viruses that contain only the rescued genome.

Following selection for viruses not carrying helper viral sequences,stocks of modified Paramyxoviridae virus are generated by infection ofVero cells, followed by scraping in medium, freeze/thaw, and frozenstorage as elsewhere herein.

F. Dosage, Administration, and Pharmaceutical Formulation

For in vivo treatments, a recombinant Paramyxoviridae virus according tothe invention is administered to the patient, preferably in abiologically compatible solution or a pharmaceutically acceptabledelivery vehicle, by ingestion, injection, inhalation or any number ofother methods. The dosages administered will vary from patient topatient; a “therapeutically effective dose” will be determined by thelevel of enhancement of function of the transferred genetic materialbalanced against any risk or deleterious side effects. Monitoring levelsof gene introduction, gene expression and/or the presence or levels ofthe encoded product are used selecting and optimize the dosagesadministered. In one embodiment, a composition including a recombinantvirus will be administered in a single dose in the range of 10³-10¹²pfus.

Ex vivo treatment is also contemplated within the present invention.Thus, in addition to direct administration of virus to patients or totheir tumors, in one embodiment, therapeutic virus is administered byintroducing virus-infected cells (preferably, but not necessarily takenfrom the same patient or tumor) to the patient or their tumor by thesame routes of administration described for virus alone. Cellpopulations, for example, tumor cells, can be removed from the patientor otherwise provided, infected with a recombinant Paramyxoviridae virusaccording to the invention, then reintroduced into the patient. Theinfection conditions ex vivo are identical to virus infection conditionsdisclosed herein; the number of cells infected ex vivo which arereintroduced into the patient are about 10⁴ to 10¹⁰ cells per day over atime course of about 1 minute to 6 hours.

In one embodiment, the course of therapy is monitored by evaluatingchanges in clinical symptoms (known in the art for each particular typeof tumor) or by direct monitoring of tumor size. Oncolytic viral therapyusing modified Paramyxoviridae viruses is effective if tumor size and/orclinical symptoms are reduced following administration of virus. Areduction in tumor size of at least 10% within a given time period, suchas one to four weeks, is desirable, and higher levels of reduction, forexample, 25%, 50% 75% and even 100% are even more desirable. In oneembodiment, in the case of a therapeutic virus modified to express amarker, the level of viral expression, and therefore the degree of viralactivity, is monitored by assays of appropriate body fluids.Alternatively, a tissue biopsy is performed in order to observesyncytium formation via direct visualization. A composition according tothe invention also is determined to be useful according to treatmentmethods of the invention where syncytium formation is observed to theextent that multinucleate areas of cytoplasm are observed in a tissuebiopsy during the course of treatment.

In another embodiment of the invention, dosing is repeated. Repeatdosing is indicated in cases in which observations of clinical symptomsor tumor size or monitoring assays indicate either that the tumor hasstopped shrinking or that the degree of viral activity is decliningwhile the tumor is still present. Repeat doses (using the same, orfurther modified virus) may be administered by the same route asinitially used or by another route.

Suitable pharmaceutical formulations, in part, depend upon the use orthe route of entry, for example oral, transdermal, or by injection. Suchforms should not prevent the composition or formulation to reach atarget cell (i.e., a cell to which the virus is desired to be deliveredto). For example, pharmacological compositions injected into the bloodstream should be soluble. Other factors are known in the art, andinclude considerations such as toxicity and forms which prevent thecomposition or formulation from exerting its effect.

EXAMPLE 1 Paramyxoviridae Virus Expressing a Marker Polypeptide

Two different MV cDNA clones were generated that express C-peptidelinked to MV H protein via either a furin-cleavable or anon-furin-cleavable linker sequence. Initially, C-peptide linked to thecleavable and non-cleavable linkers were cloned into a pCG vector as aC-terminal extension of H protein to generate vectors pCGHFurCP andpCGHG4SCP, respectively (See FIG. 1 for a schematic diagram).

To produce pCGHFurCP, primers Fur CP1.Nf and SfurinCP were used in a PCRreaction with pro-insulin template (ATCC) for 18 cycles at 94° C. 1 min,55° C. 1 min and 72° C. for 1 min. The resulting PCR fragment, taggedwith Sfi-CP-Not was gel purified and digested with SfiI and NotIrestriction enzymes. The pCGH backbone pCGH EGFr- was also digested withSfiI and NotI, and ligated with the prepared insert. Ligation reactionswere transformed into competent E. coli, and resulting colonies screenedby PCR for the presence of C-peptide insert. Successful transformantswere grown to large scale and the DNA isolated. FurCP1.Nf ttt tcc ttttgc ggc cgc ttt cat caa cgc ttc tgc agg gac ccc tc SFurinCP gtc cat gcggcc cag ccg gcc CGA TTA AAG AGA gag gca gag gac ctg V H A A Q P A R L KR E A E D L cag gtg gg Q V

To produce pCGHG4SCP, primers G4SCP1.Nf and SG4SCP.b were used in a PCRreaction as described above. G4SCP1.Nf ttt tcc ttt tgc ggc cgc ttt catcat caa cgc ttc tgc agg gac ccc tc SG4SCP.b gtc cat gcg gcc cag ccg gccGGT GGA GGC GGT TCA gag gca gag gac V H A A Q P A G G G G S E A E D ctgcag gtg gg L Q V

The DNA fragments encoding H-linker-CP were digested from the pCGconstructs using enzymes PacI and SpeI, and transferred into the MV cDNAclone p(+)MV-Nse by ligation with PacI/SpeI digested DNA. The resultingfull length cDNA constructs encoding H linked to C-peptide via a furincleavable or non-cleavable sequence were named p(+)MV-HFurCP andp(+)MV-HG4SCP, respectively.

To recover replicating chimeric measles viruses, 5 μg of each constructwas transfected into 293-3-46 cells with 5 ng pEMCL (expressing the MVpolymerase, L) using calcium phosphate. Following transfection, thecells were overlaid onto Vero cells which were observed for theformation of syncytium. Syncytia were picked and transferred to freshcells to expand the virus stock, which was then prepared by freeze/thawand titered using a conventional TCID50 assay.

To check whether the viruses produce C-peptide, T75 tissue cultureflasks with confluent monolayers of Vero cells were infected with 10⁵pfu of virus for 2 h at 37° C. The media was replaced with 5 ml of 5%FCS-DMEM for 16 h and then harvested for C-peptide assay. Results aretabulated below, and show that the CP expressed by the furin sensitivelinker is secreted into the extracellular media. MV Clone C-Peptide inMedium MV-NSe <33 pM C-peptide MV-H/FurCP 7000 pM MV-H/G4SCP 74 pM

EXAMPLE 2 Paramyxoviridae Viruses with Enhanced Fusogenicity

Fusogenicity of Paramyxoviridae viruses may be enhanced by modificationof the F, H or M proteins.

A. Modification of Fusogenicity by Modification of Measles Virus FProtein

F protein cytoplasmic tail mutations have been introduced to plasmidsencoding full length measles virus genomic RNA (e.g., p(+)MV, Radecke etal., supra) as follows. Plasmids peFHLP, peFHLF and peFHLI (described inSchmid et al., 1992, Virology 188: 910) and plasmid peF(cSeV)HL wereused as starting material for generation of full length measles virusplasmids encoding the following F mutants, respectively: mutant Fc+28,resulting from a stop codon mutation, comprises 28 amino acids ofextraneous sequence appended to the C-terminus of the wild-type Fprotein sequence (Fc+28 also has an additional Glu relative to theEdmonston B strain of the virus at position 27 relative to thetransmembrane domain); mutant FcΔ16 lacks the 16 C-terminal amino acidsof the F cytoplasmic tail (remaining sequence of the cytoplasmic tail isRGRCNKKGEQaGMSRPG, where the lower case “a” also differs from thesequence of the Edmonston B strain of measles virus); mutant FcΔ24 lacks24 C-terminal amino acids relative to the wild-type virus (remainingsequence of the cytoplasmic tail is RGRCNKKGE); and mutant FcSeV has themeasles virus F protein cytoplasmic tail replaced by the F cytoplasmictail from Sendai virus. Plasmid pcF(cSeV)HL was generated by subcloninga PstI-PacI PCR fragment encoding the SeV F cytoplasmic tail into peFHL.PCR was performed with pGem4-SVF₀ (described in Vidal et al., 1989, J.Virol. 63: 892) as the template and primers5′-AAAACTGCAGACTCAAAGGTCAATGC-3′ and5′-CCCTTAATTAATATACAGATCTCAACGGAT-3′.

The genomic measles virus plasmids comprising the F cytoplasmic tailmutations were generated by three-way ligations of a NarI-PacI fragmentcarrying the mutated F gene coding region from plasmid peFHLP, peFHLF,peFHLI or peF(cSeV)HL, respectively, with a PacI-SacII fragment and aSacII-NarI fragment of p(+)MV. All full length plasmids coding formutated antigenomic RNA conform to the rule of six.

Infectious viruses comprising the F protein mutations were rescued fromtransfected cells according to the method of Radecke et al. (1995,supra) and used to infect Vero cells at an MOI of 3. Cytopathic effects,including syncytia formation, were monitored over time by microscopicexamination. All F mutant viruses tried produced syncytia. Virusesencoding the F mutants Fc+28, and FcΔ24 produced syncytia that grewfaster than those induced by infection with wild-type virus.

B. Modification of Fusogenicity by Modification of Measles Virus HProtein

H protein mutant plasmids peHcΔ8 (lacking amino-terminal amino acids2-9), peHcΔ14 (lacking amino terminal amino acids 3-16), and peHcΔ24(lacking amino-terminal amino acids 2-25) were constructed by subcloninga ClaI-EcoRI PCR fragment into peH5. peH5 is a shuttle vector forsubcloning into the full-length p(+)MV (Cathomen et al., J. Virol.,supra). peH5 contains a single ClaI site in the 5′ untranslated regionof the H protein and a single EcoRI site at the border between thetransmembrane domain and the ectodomain, both sites introduced by silentmutations. PCR was performed with peH5 DNA as template and forwardprimers 5′-CCATCGATAATGGCCTTCTACAAAGATAACC-3′ (peHcΔ8),5′-CCATCGATAATGAGCCATCCCAAGGGAAGTAGG-3′ (peHcΔ14), and5-CCATCGATAATGAACAGAGAACATCTTATGATT-3′ (peHcΔ24). The reverse primerannealed downstream of the H protein coding region in the plasmid.

To construct the H mutant in which the measles virus cytoplasmic tailwas replaced with the Sendai virus H cytoplasmic tail, fusion PCR wasperformed. The SeV H-tail encoding region was amplified from thepGem4-SVHN plasmid template (described by Vidal et al., supra) withprimers 5′-CCATCGATAATCATGGATGGTGATAGGGG-3′ and5′-GCAAAACATAAGGGGTGTCAACTTTACTTGA-3′. The primer5′-GACACCCCTTATGTTTTGCTGGC-3′ and a primer annealing downstream of theregion coding for the H transmembrane domain were used to amplify the MVH transmembrane encoding region. In the fusion step, the isolated PCRfragments with an overlapping sequence of 19 nucleotides (underlined)were mixed and amplified with the external primers. The resultingfragment was digested with ClaI and EcoRI and then subcloned into peH5.

The various peH5 subclones carrying the H mutant coding sequences weredigested with ClaI and EcoRI and the H mutant fragments inserted intop(+)MV. Infectious viruses comprising the H protein mutations wererescued from transfected cells according to the method of Radecke et al.(1995, supra) and used to infect Vero cells at an MOI of 3. Cytopathiceffects, including syncytia formation, were monitored over time bymicroscopic examination. Viruses encoding the H mutants HcΔ8, HcΔ14, andHcSeV produced syncytia, while HcΔ24 did not. HcΔ8 and HcΔ14 producedextensive syncytia (greater than 90% of nuclei in syncytia).

C. Combination of F and H Mutations.

Measles viruses encoding both mutated F and mutated H proteins weregenerated by replacing the PacI-SacII fragment encoding the H protein ofthe p(+)MV-Fc+28, p(+)MV-FcΔ24 and p(+)MV-FcSeV plasmids, with thePacI-SacII fragment encoding HcΔ14.

Double mutant viruses were rescued from cells transfected with thegenomic plasmids as above and used to infect Vero cell monolayers. Alldouble mutants tested induced cell fusion more rapidly and moreextensively than wild-type virus. At 36 hours post-infection, almost allof the cells infected with double mutant viruses were fused in largesyncytia (>100 nuclei per syncytium), as compared to nearly no syncytiaat the same time in cells infected with wild-type virus.

D. Modification of Measles Virus Fusogenicity by Alteration of M Protein

An M protein-less mutant of measles virus was constructed by removal ofa 960 nucleotide BglII-BclI fragment containing the M protein codingsequences from the p(+)MV plasmid, to generate p(+)MV- M (Cathomen etal., EMBO J, supra). The M-less genomic plasmid was transfected into293-3-46 helper cells (Radecke et al., 1995, supra). After 3-5 days,syncytia were observed, indicating rescue of infectivity.

Infection of Vero cell monolayers with M-less measles virus showed thatat any given time after infection, the MV-ΔM syncytia were larger thanthose induced by wild-type reference virus. Quantitation of the extentof cell fusion after infection with a MOI of 0.01 revealed that after 4days approximately 75% of the nuclei of an MV- M-infected cell monolayerwere in syncytia, compared with approximately 25% of the nuclei of MVinfected cells. After 6 days, >90% of the cells in both cultures wereinvolved in syncytia, and cell mortality was substantial. These dataindicate that the M-less virus is more efficient than the standard MV atinducing cell-cell fusion.

EXAMPLE 3 Paramyxoviridae Virus Expressing a Cytokine

A Paramyxoviridae virus modified to express IL-12 is constructed asfollows. IL-12 is comprised of two subunits, p35 and p40. The sequencesfor both subunits are encoded by plasmid pBsIL-12, separated by an IRESfrom encephalomyocarditis virus (plasmid is described in Hemmi et al.,1998, Hum. Gene Ther.). pBsIL-12 was digested with NotI/XhoI andblunted, and the resulting 2306 bp fragment was ligated into the NruIsite of peFHaigrL (described in Singh and Billeter, 1999, J. Gen. Virol.80: 101) to obtain peFHLIL-12. A PacI-Spe fragment of peFHLIL-12 wasplaced in p(+)MVNSe (containing the antigenomic MV tag-Edmonston Bsequence, slightly modified from p(+)MV (Radecke et al., 1995, supra) toexhibit unique NarI and SpeI sites) to obtain plasmid p(+)MVIL-12. Thislocates the IL-12 sequences between the H and L coding regions as anadditional cistron.

The MV-IL12 virus is rescued using the method of Radecke et al. (1995,supra). Syncytia developing in the rescue cultures are picked and usedto infect Vero cell monolayers to expand the virus.

Expression of IL-12 directed by the virus is demonstrated by Westernblotting protein from infected cells using antibodies specific forIL-12. IL-12 produced by virus-infected cells is tested by monitoringinduction of IFN-γ secretion from peripheral blood mononuclear cells(PBMCs); see Singh and Billeter, 1999, J. Gen. Virol. 101.

EXAMPLE 4 Paramyxoviridae Virus Modified to Alter Protease Sensitivity

A Paramyxoviridae virus dependent on trypsin cleavage, rather than furincleavage, for activation of infectivity is generated as follows.

A. Modification of Measles Vines F Protein

Measles virus F protein was modified by changing the arginine atposition 109 and the lysine at position 111 to asparagine. Cloning ofthe viral glycoprotein (H and F protein) genes into the expressionvector pCG under the control of the CMV early promoter has beendescribed by Cathomen et al., 1995, supra. The F cleavage mutant(pCG-Fcm) with substitutions in the furin recognition motif was preparedby introduction of site-specific mutations (underlined) with thecomplementary primers Fcm1 (5′-GCTTCAAGTAGGAACCACAACAGATTTGCGGG-3′) andFcm2 (5′-CCCGCAAATCTGTTGTGGTTCCTACTTGAAGC-3′) into the double-strandedpCG-F plasmid using the QuikChange Site-Directed Mutagenesis Kit(Stratagene). After dideoxy sequencing of the complete F gene, themutagenized plasmid was used for transfection of 293 and Vero cells.

For the generation of recombinant MV, a derivative (p(+)MVNSe; Singh etal., 1999, J. Virol. 73: 4823) of the cDNA clone containing the fulllength MV Edmonston B based genome described by Radecke et al. (1995,supra) was used. To construct a full length MV genome with a mutated Fprotein, the F protein was mutagenized in the shuttle vector peF1(Radecke et al., 1995) using the primer pair Fcm1 and Fcm2. A NarI-PacIfragment containing the F gene with the mutated cleavage site wassubcloned into p(+)MVNSe and completely sequenced by the dideoxy methodusing an automatic sequencer (Perkin Elmer). The standard cDNA clone(p(+)MVNSe) as well as the mutated clone (p(+)MV-Fcm) were used togenerate recombinant MV.

B. Virus Rescue and Preparation of Recombinant Virus Stocks

Transfection and rescue of MV were performed mainly as described byRadecke et al., 1995, supra). Briefly, 293-3-46 helper cells mediatingboth artificial T7 transcription and NP and P functions were transfectedwith 8 μg either p(+)MVNSe or p(+)MV-Fcm in the presence of 5 ng ofplasmid encoding the MV polymerase (pEMC-La).

At two days posttransfection, cells were expanded. To induce syncytiumformation in p(+)MV-Fcm transfected cells, cells were washed andactivated for 2 h at 37° C. with DMEM/trypsin. After activation, cellgrowth was allowed to proceed in DMEM supplemented with 10% FCS. At 4 dposttransfection, transfected cells were scraped in OptiMEM (GIBCO-BRL)with trypsin (1 μg/ml) and adsorbed to Vero cell monolayers. Afterwashing, infected Vero cells were kept in DMEM/trypsin because theytolerate a trypsin concentration up to 1.2 μg per ml of medium withoutdetaching (in contrast to 293 cells). At 5 d posttransfection, multiplesyncytia indicated the successful rescue of p(+)MVNSe (MV-Edm). Firstsyncytia in the p(+)MW-Fcm rescue appeared 7 to 8 d posttransfection.

Single syncytia were picked for infection of a Vero cell monolayer inthe presence of trypsin. When the cytopathic effect (CPE) reached 90%,the cells were scraped into 1 ml of the cell culture medium andsubjected to two rounds of freezing and thawing. The clearedsupernatants were considered as “plaque purified” recombinant virus(MV-Edm, MV-Fcm). To produce virus stocks, cleared supernatants weretaken to infect subconfluent Vero cell monolayers. During infection at33° C., the cells were kept in DMEM/trypsin. Infected cells showing 90to 100% CPE were scraped into the medium, frozen and thawed, aliquottedand stored at −80° C. Infectivity was determined by 50% end-pointdilution assay (TCID50, see Cathomen et al., 1998, J. Virol., supra).

C. Fusion Assay

Because of the sensitivity of 293 cells to trypsin, fusion activity wasanalyzed in transiently transfected or infected Vero cells,respectively. For syncytium formation, Vero cells were cotransfectedwith standard or mutant F gene (pCG-F, pCG-Fcm) and standard H proteingene (pCG-H). At 16 h posttransfection, the cells were washed twice withPBS to remove the FCS containing medium and were further incubated withDMEM. To one monolayer from each set of duplicate samples TPCK-treatedtrypsin (Sigma) at a concentration of 1 μg per ml media was added. At 24h posttransfection, the transiently expressing cells were fixed withethanol and stained with 1:10 diluted Giemsa's staining solution (Merck,Darmstadt, Germany). To analyze the biological activity of recombinantMV with a mutated F protein, subconfluent Vero cells were infected withstandard (MV-Edm) or mutant MV (MV-Fcm) at a multiplicity of infection(MOI) of 0.01-0.1. The infected cells were cultivated at 37° C. in DMEMin the absence or presence of 1 μg trypsin per ml media. At 24 hpostinfection, the infected cells were fixed and stained as described.

D. Immunostaining

Subconfluent Vero cells (1×105 cells) were grown on coverslips andinfected with MV-Fcm at an MOI of 5 for 2 h at 37° C. The cells wereintensively washed with PBS overlaid with DMEM or DMEM/trypsin andincubated for 28 h at 33° C. to allow one step growth in the presence orabsence of trypsin. To quantify the amount of infected cells,immunostaining was performed. After fixation and permeabilization at−20° C. with methanol/acetone (1:1), MV positive cells were detectedwith a polyclonal rabbit antiserum raised against purified MV and aFITC-labeled goat anti-rabbit IgG (DAKO, Denmark). The samples weremounted in mowiol and 10% triethylendiamine. For quantification ofinfectious cell-free virus, the cell supernatant was collected beforeimmunostaining of the infected cells (28 h p.i.). The supernatant (300μl) was directly used to infect fresh Vero cells grown to subconfluencyon coverslips.

To activate cell-free virus grown in the absence of trypsin, 1 μgTPCK-treated trypsin per ml supernatant was added. As a control, 300 μlof the supernatant without trypsin addition was used for infection.Virus adsorption in the absence or presence of trypsin was allowed toproceed for 4 h at 37° C. Then, the cells were washed several times withPBS overlaid with DMEM and incubated at 33° C., To quantify the amountof infected cells, the cells were fixed at 42 h p.i. and immunostainingwas performed as described.

E. Mice Infections

The Ifnartm-CD46Ge mice used in this study have a targeted mutation (tm)inactivating the interferon receptor type I (Ifnar). Since a yeastartificial chromosome covering about 400 kilobases of human genomesurrounding the CD46 gene (CD46Ge) was transferred to mice, theseanimals express CD46 with human-like tissue specificity (Mrkic et al.,1998, J. Virol. 72: 7420). Age-matched mice were used for infections atthe age of 6 to 7 weeks. For intranasal inoculation a total volume of 50μl of appropriate virus stocks was administered into both nares.Intracerebral inoculations were done along the midline by using a27-gauge needle. The inoculum consisted of 30 μl of stock virus dilutedin PBS.

F. Histology and In Situ Hybridization Assay

Assays were basically performed as described previously (Mrkic et al.,1998). Briefly, mice were euthanized with C022, the lungs were removedand fixed in 4% PBS-buffered formaldehyde. Paraffin-embedded tissueswere cut at 2-3

m sections. For general histological analysis the sections were stainedwith hematoxylin/eosin (HE) staining solution. Detection of MV N mRNA insitu was performed with a digoxigenin (DIG)-labeled N RNA probe (30 pg/

g) followed by immunological staining with a DIG-nucleic acid detectionkit (Boehringer Mannheim). The sections were counterstained withhematoxylin solution.

Transient transfection of the recombinant expression plasmids pCG-F andpCG-Fcm into 293 cells was used to monitor the effect of the alterationsto the furin cleavage site on the cleavage of the protein. Western blotanalysis of transfected lysates under non-reducing conditions indicatedthat the alteration of the furin cleavage site resulted in complete lossof cleavage of the F0 protein in the absence of trypsin.

The effect of the furin cleavage site modification on cell-cell fusionwas tested by transfecting either wild-type or mutant F plasmids intoVero cells with a wild-type H gene expression plasmid (pCG-H). Toactivate uncleaved F protein on the cell surface, 1 ug of trypsin per mlwas added to one of each set of duplicate samples at 16 hoursposttransfection. Cells were fixed and stained at 24 hoursposttransfection. Cotransfection of standard MV glycoproteins (H+F)induced syncytium formation in the absence and presence of trypsin.Coexpression of standard H protein and mutant F protein (H+Fcm) onlyinduced cell fusion in the presence of trypsin, indicating that mutatedF protein was transported to the cell surface where it could bebiologically activated by trypsin cleavage to cause syncytium formation.

In order to rescue Fcm mutated virus, the Radecke method was used,except that trypsin was required. Therefore, the modification to thefurin cleavage site renders the virus dependent on trypsin foractivation of infectivity.

Vero cells infected with MV-Edm (wild-type strain) or MV-Fcm at amultiplicity of infection of 0.01 to 0.1 in the presence and absence oftrypsin were analyzed by Western blot at 24 h postinfection. About 95%of F protein was found as the F1 subunit in cells infected with standardor wild-type virus, regardless of whether trypsin was included in themedium. In contrast, 100% of the F protein migrated as precursor F0 whenthe mutant virus was used in the absence of trypsin. Only when mutant Fprotein was synthesized in infected cells cultivated in the presence oftrypsin was a significant amount of the cleavage product F1 detected.This indicates that virus-derived mutant F protein, in contrast tostandard F protein, was not susceptible to intracellular cleavage bytrypsin. For these experiments, control probing of protein blots withanti-H antibodies indicated no difference in H expression between cellsinfected with mutant or wild-type virus, indicating that infection byMV-Edm and MV-Fcm was comparable and that H protein expression was notinfluenced by trypsin addition during 24 h of infection.

Similarly, cell fusion in infected cultures was not observed unlesstrypsin was included in the medium. In order to test for cell fusionactivity of standard MV versus MV-Fcm, Vero cells were infected andcultivated in the absence and presence of trypsin and tested forsyncytium formation at 24 h postinfection. MV-Edm cells showedtrypsin-independent cell fusion activity. Cells infected with MV-Fcm didnot show any syncytium formation when cultivated in the absence oftrypsin, but cultivation in the presence of trypsin resulted in cellfusion activity.

As both MV-Fcm and MV-Edm are released from infected cells and do notsignificantly differ in the protein composition of viral particles, itwas important to demonstrate that MV-Fcm grown in the absence of trypsinis actually non-infectious and activation of cell-free virus completelydepends upon extracellular proteases. Subconfluent Vero cells wereinfected at an MOI of 5 for 28 h in the presence or absence of trypsin.To monitor the efficiency of infection, the infected cells were stainedwith an antiserum raised against purified MV. As expected, all cellswere MV positive, and cells infected in the presence of trypsin showedalmost complete fusion, whereas cells infected in the absence of trypsindid not show any syncytium formation.

To test if infectious virions were released into the culture media,supernatants were used to infect fresh Vero cells. The supernatant ofMV-Fcm grown without trypsin was either used untreated or complementedwith trypsin to a final concentration of 1 ug/ml. The supernatants wereallowed to adsorb for 4 h at 37oC and the removed by extensive washings.Since further infection was performed without trypsin, no virus spreadoccurred, and the amount of MV-infected cells directly reflects theamount of infectious particles. At 42 h postinfection, MV positive cellswere detected by immunostaining. No MV-Fcm positive cell was detectedafter infection with supernatant of cells infected in the absence oftrypsin. Addition of trypsin to this “noninfectious” supernatant duringvirus adsorption resulted in the infection of about 30% of the cells,clearly demonstrating that the supernatant contained cell-free MV-Fcmparticles that could be activated by trypsin.

To examine the effect of the furin to trypsin alteration of MV-Fcmcleavage in vivo, a mouse model was used. A group of eightIfnartm-CD46Ge mice were infected intranasally with 3×105 PFU to studythe pathogenic effects of MV-Fcm replication after uptake through therespiratory route. As a control, six mice were infected with MV-Edm. Thelungs were removed for histological analysis and in situ hybridizationat 4 days postinfection when high levels of standard virus replicationwas observed. Standard MV-Edm infection causes acute lung inflammation,extensive hyperemia and diffuse hemorrhage in large areas of the lung(Mrkic et al., 1998, supra). Although less pronounced than in standardvirus infection, mutant MV also caused pathological effects.

MV-Fcm infected mice revealed an increased cellular density andinfiltration of inflammatory cells, particularly in the perivascularregions. To demonstrate virus replication, MV-infected cells weredetected by MV N-specific in situ hybridization assay. After standardvirus infection, MV positive cells were mainly found close by or in thealveolar epithelium, often in cell groups indicating virus spread bycell-cell fusion. In MV-Fcm infected mice, single virus-positive cellswere distributed all over the whole lung tissue, but the majority wasfound in the alveolar walls. Cells of the bronchiolar epithelium wereoccasionally infected. No virus-positive cells were detected, indicatingthat virus spread by cell-cell fusion probably did not occur. The amountof virus-positive cells was rather small, suggesting that MV-Fcmreplication was not as efficient as replication of parental MV-Edm.However, MV-Fcm was able to induce lymphatic infiltration in the lung.These findings support the notion that the F protein is activated in thelung by secreted proteases, resulting in productive infection in thelung.

The sensitivity of IfnarTM-CD46Ge mice to intracerebral infection withMV-Fcm was also tested. When infected with MV-Edm, 5 of 6 animals showedclinical signs of neural disease and died within one week afterinfection. In contrast, all mice infected with MV-Fcm survived and didnot develop any signs of disease. Thus, MV-Fcm was not pathogenic inmice when inoculated in the brain, probably due to the lack ofactivating proteases in this organ.

EXAMPLE 5 Generation of a Library Containing Partially RamdomizedSequence Between F1 and F2 (F Library)

In this example, the standard MV F cleavage signal RHKR, recognised byfurin, is mutated using random primers to generate a library withpotentially novel cleavage specificities. To avoid restoring furinsensitivity, primers are designed such that positions 1,2,3 cannot bearginine or lysine. Furin Cleavage  ▾ 1 2 3 4 5 6 7 MV F0 R H K R F A GFurin sensitive site Beginning of F1 polypeptide  ▴

Arginine is coded by (single amino acid codes)

-   -   AGG    -   AGA    -   CGG    -   CGA    -   CGC    -   CGT

Lysine is coded by:

-   -   AAG    -   AAA

The possible permutations of bases (as a result possible amino acids)are as shown: Furin cleavage ▾ 1 2 3 4 5 6 7 R H K R F A G L I A Primer:XAT XAT XAT XXX TTC GCA GGT XCC XCC XCC XXX TTA ATA GCT XTX XTX XTX XXX

To eliminate R or K from positions 1,2,3, the possible permutation ofbases are as shown above (where X=Any base). Thus for position 1, thefirst base can be A, T, C or G), 2nd base can be A, C or T and 3rd basecan be T or C.

To make the library of F cleavage mutants, a PCR fragment of measles Fis first generated. Through introduction of BamHI sites in the PCRprimers, the PCR fragment is made flanked by restriction sites forBamH1, facilitating cloning into the transfer vector (containingretroviral LTR elements) pMFG. pMFG is derived from pMFG nlsLac Z, byremoval of the nlsLacZ fragment by BamH1 digest.

After introduction to the transfer vector, the plasmid library isco-transfected with retroviral gag-pol and envelope expression plasmidsinto AM12 viral producer cells. The retroviral library is harvested andused to infect Vero cells which have been previously transfected withpCGH plasmid. Infection may be performed in the presence or absence ofprotease/protease inhibitors. Syncytia are picked, or balls ofmultinucleated syncytia which have floated into the supernatant arepicked and sorted according to size of the floating balls. The Ffragment is amplified by PCR from picked syncytia and sequenced todetermine the linker sequence between F1 and F2.

EXAMPLE 6 Attenuated Measles Virus Targeting Specificity for CD38

A. Generation of Recombinant Viruses Displaying Single Chain AntibodyFragments

In one embodiment of the invention, the targeting specificity of anattenuated virus was altered by displaying a single chain antibody(ScFv) on the surface of the virus. In this embodiment, cDNAs coding forscFvs against human lymphocyte differentiation antigens CD38 and CD52were ligated into a full length infectious clone of MV-Edm (FIG. 5). Thegenes were inserted as in-frame fusions linked to the C-terminal codonof the H glycoprotein through a linker sequence encoding an IEGR (singleamino acid coade) factor Xa protease cleavage signal. MV-Edm recombinantviruses were recovered from these constructs and were amplified in CD46receptor-positive Vero cells in which they replicated as efficiently asunmodified MV-Edm. Correct expression of the scFV domains was confirmedon immunoblots of cell lysates of infected Vero cells, and probed withan antibody against the H glycoprotein (FIGS. 6A-D).

B. Targeted Cell Entry and Cytotoxicity of MV-Edm Displaying an Antibodyto CD38

To determine whether the displayed anti-CD38 scFv could redirect virusattachment and entry, MV-Edm and scFv-displaying MV-Edm were titrated onCD46-negative Chinese Hamster Ovary (CHO) cells expressing human CD38cells and control CHO cells. Infectious centers, defined asmultinucleated syncytia containing more than 20 nucleic were counted 36hours after invention. MV-EdM displaying an scFV against CD38 readilyinfected CD38-expressing CHO cells but was unable to infect unmodifiedCHO cells or CHO cells stably expressing human EGF-receptor (FIGS.6A-D). Unmodified and CD52 scFv-displaying MV-Edm were not infectious onthe CHO cells, irrespective of CD38 status (FIGS. 6A-D). Cleavage of theanti-CD38 antibody protion from the MV-Edm recombinant virus usingFactor Xa protease ablated the infectivity of anti-CD38 scFv displayingvirus on CD38-expressing CHO cells (FIGS. 6A-7). The virus was stillable to infect CD46-expressing cells after protease cleavage, indicatingthat the functional integrity of the underlying H glycoprotein was notcompromised by exposure to Factor Xa protease (FIG. 7).

EXAMPLE 7 Reombinant Measles Virus with Targeting Specificity for CEA

To redirect the tropism of measles virus (MV) to a targeted cellpopulation, a single chain antibody (scAb) specific for the tumorassociated carcinoembryonic antigen (CEA) was displayed on the viralhemagglutinin (H). The targeted antigen, CEA, is highly over-expressedon the surface of a number of cancerous cells, particularly ofcolorectal, gastric, lung, pancreatic and breast carcinomas. Itsexpression in normal adult tissue is restricted to selected epithelialcells, and the anti-CEA (αCEA) scAb used, MFE-23, has littlecross-reactivity to non-malignant human tissue.

Constructs were generated expressing three forms of the αCEA MFE-23 scAbas C-terminal fusions of H (FIG. 8A). The scAb forms differed in thelength of the linker separating the VH and VL domains, and weredesignated zero (0), short (S) and long (L), corresponding to linkerlengths of 0, 6 and 16 amino acids, respectively. In each construct, theC-terminus of H was separated from the scAb by an 8 amino acid spacerincluding a Factor Xa cleavage site to facilitate removal of thedisplayed ligand. The chimeric HαCEA proteins were designated HX0, HXSand HXL accordingly.

To generate constructs in which the displayed scAb could beproteolytically cleaved away from the H protein, each scAb was PCRamplified from the pCG constructs using primers which generated a BssHIIsite upstream of the scAb cDNA, and which maintained the NotI site atthe 3′ end. These PCR products were digested with BssHII and NotI,purified and ligated with BssHII/NotI digested pCG-H—X-RGD, such thatthe RGD peptide in this construct was replaced with the scAb which wasthus positioned 3′ to the Factor Xa cleavage site (see FIG. 8A).Sequence analysis revealed all clones except the pCG-H—X—O-CEA to becorrect.

To test the ability of these chimeric H proteins to mediate fusion, andto test whether any re-directed the fusion function of H to CEA, eachconstruct was co-transfected using Superfect (Qiagen) with the plasmidpCG-F encoding a functional MV F protein, into HeLa cells and HeLa cellsoverexpressing CEA. Cells were observed for syncytial formation, andnumbers of syncytia were scored for each H-scAb construct (Table 2). TheH protein expressing the long linker form of the scAb preferentiallymediated fusion on HeLa-CEA cells (FIGS. 9A-B). TABLE 2 Ability OfChimeric H Proteins To Mediate Fusion On CEA Negative And Positive CellLines. HeLa or Vero HeLa-CEA H5 + F +++++ +++++ H5-O-CEA + F − −H5-S-CEA or H5-X-S-CEA + F − + H5-L-CEA or H5-X-L-CEA + F ++ ++++

To generate recombinant MV expressing a scAb directed against CEA, thecDNA encoding H—X-L-CEA was digested from the pCG construct using theenzymes PacI and SpeI, purified and ligated with PacI/SpeI digestedp(+)MV-NSe, to generate the construct p(+)MV-NSe-H/X-L-CEA. Virus wasrecovered by calcium phosphate co-transfection of this plasmid alongwith pEMC-La, encoding the MV polymerase L, into 293-3-46 cells stablyexpressing T7 polymerase and the MV proteins N and P. Following overlayof these transfected cells onto Vero cells, observed syncytia werepicked and the virus expanded by passage on fresh Vero cells.

All proteins were stable, appeared properly folded, and were transportedto the cell surface, but only H displaying the long linker form of scAb(HXL) was functional in supporting cell-cell fusion. HXL inducedextensive syncytia in cells expressing the normal virus receptor CD46,and also in CD46-negative cells expressing the targeted receptor, humanCEA. Replication competent MV with H substituted by HXL was recovered,and was dual tropic, replicating efficiently in both CD46-positive andCD46-negative, CEA-positive cells. Thus MV not only tolerates theaddition of a large scAb on its H protein, but can be engineered toinfect cells via a novel interaction between a displayed scAb and itstargeted receptor.

The long linker form induced extensive syncytia in both CD46-positiveand CD46-negative, CEA-positive cells. A replicating MV expressing thischimeric protein in place of H was generated. Significantly, this virusreplicated not only with the efficiency of unmodified MV inCD46-positive cells, but almost as efficiently in CD46-negative cellsexpressing CEA, which unmodified MV failed to infect.

A. Plasmid Construction and MV Recovery

cDNAs encoding the three forms of the scAb were transferred to a pCG-Hvector (17) containing a Factor Xa cleavage site 3′ to the H ORF fromretroviral expression vectors (J. Zhang, data not shown) using PCRamplification (primer sequences: 5′-GCGCGCTGGCCCAGGTG-3′ and5′-TGCGGCCGCCCGTTTC-3′, BssHII and NotI sites underlined). For detectionpurposes, an amino-terminal Flag tag (DYKDDDDK) was inserted downstreamof the ATG start codon of each H construct. DNA sequencing confirmed theintegrity of all constructs. The cDNA encoding HXL was transferred fromthe pCG construct into a molecular clone of MV-Edmonston, p(+)MV-NSe(18). Virus was rescued as previously described (19).

B. Cell Culture and Transfection

Vero, HeLa, HeLa-CEA and MC38 cells were maintained in 10% FCS/DMEM.MC38-CEA (clone 2) cells (Robbins, et al. Cancer res. 51: 3657-3662)were maintained in 10% FCS/DMEM containing 0.5% G418. Cells weretransiently transfected using Superfect (Qiagen) and analyzed 18-24hours post-transfection. For syncytia formation assays, target cells(5×105/well, 35 mm wells) were co-transfected in duplicate with 1.5 μgDNA encoding F and 1.5 μg DNA encoding the appropriate H protein.Syncytia in 20 representative fields (20% of a 35 mm well) were countedat indicated times and the number of syncytia per well calculated.

C. MV Stocks and Infection

Preparation of MV stocks, virus propagation, purification and titrationwere performed as previously described (Radecke, et al., EMBO 14:5773-5784). For infection, the appropriate MOI of cell-associated viruswas adsorbed for 2 hours with target cells (5×105/well, 35 mm wells).For proteolytic digestion of the displayed domain, viruses in clarifiedcell extracts (MOI of 1) were pretreated with 10 μg/ml Factor Xa (NewEngland Biolabs) for 2 hours at 23° C. prior to adsorption. For antibodyadsorption of cell surface CEA, cells were pretreated with 10

g/ml COL1 (LabVision Corp.) for 2 hours prior to infection with an MOIof 1. For both treatments, levels were maintained at 10 μg/ml byreplacing with fresh media containing inhibitor every 12 hours.

D. Western, Pulse Chase and H Dimerization Analyses.

Western analysis of MV proteins from transfected or infected cells,pulse chase analysis of H from transfected Vero cells and analysis ofhetero- and homotypic H dimerization, was performed as previouslydescribed (Plemper, et al., J. Virol. 72: 1224-34, 1998). HαCEA proteinswere detected or immunoprecipitated using an αFlag Ab (M2, Sigma) and MVparticles were analysed using an MV-specific goat antiserum.

E. Facs Analysis

Target cells (5×105/reaction) were incubated on ice in PBS/FCS/azide for30 minutes, then with primary Ab for 1 hour at 4oC Cells were washed,incubated with secondary Ab, repeatedly washed, fixed in 0.4%paraformaldehyde and analysed using a Becton-Dickinson FACSCalibur andCellQuest software. For detection of virus binding to the cell surface,cells were preincubated with virus at an MOI of 3 for 2 hours at 4° C.The 11/88 mAb (Schneider-Schaulies, et. al.) J. Virol. 69: 2248-56,1995)) was used to detect surface CD46, COL1 mAb to detect CEA, and mAb129 (Sheshberadaran, et al. Virology 128: 341-353, 1983) to detectsurface H and virus bound to the cell surface. All primary antibodieswere realized using an αmouse-FITC conjugate (Jackson).

F. Expression, Stability, Oligomerisation and Cell Surface Localisationof Chimeric HαCEA Proteins

Expression of HX0, HXS and HXL proteins at the expected molecular weightin all cell lines used was confirmed by Western blot analysis (shown inFIG. 8B for Vero cells, data not shown for HeLa, HeLa-CEA, MC38 andMC38-CEA cells). Furthermore, pulse chase analyses demonstrated asimilar stability of expression for the chimeric H

CEA proteins as for unmodified H, with half lives for all proteins ofgreater than 3 hours (FIG. 13A).

The H-like conformation of the HXL protein was verified by its abilityto form covalently linked dimers with itself and with unmodified H.Following co-transfection of Flag-tagged HXL with unmodified, untaggedH, or with empty plasmid or Flag-tagged H for control, cells weremetabolically labelled. Using an αFlag Ab, tagged proteins and anyinteracting untagged H proteins were immunoprecipitated, and thedimerization status was analysed by gel electrophoresis undernon-reducing conditions (FIG. 13B).

Both homotypic dimers of HXL/HXL and heterotypic dimers of HXL/H wereidentified. Under conditions in which both types of complex could form,the heterotypic HXL/H complex predominated, suggesting dimerization ofHXL with unmodified H was more efficient than with itself. Theefficiency of both HXL/HXL and HXL/H complex formation was, however,reduced compared with that of H/H dimerization, thus display of the scAbon H reduces the efficiency of, but does not prevent, dimerization ofthe underlying H molecule.

Since the formation of covalently linked dimers is a prerequisite forefficient H transport (Plemper, et al., J. Virol. 74: 6485-6493, 2000),our data suggested that the HXL protein should be efficientlytransported. Indeed, cell surface expression of not only HXL but all H

CEA proteins was confirmed by FACS analysis of transfected cells to besimilar to that of unmodified H (FIG. 13C), indicating efficienttransport for all HαCEA proteins.

G. HXL Supports Syncytia Formation in Both CD46-Positive andCD46-Negative, CEA-Positive Cells

Although display of a scAb on MV H did not affect its proper folding ortransport, its receptor binding and fusion support functions may havebeen disrupted. We assessed the functionality of the HαCEA proteins bymeasuring syncytia formation following co-expression with MV F.

In three CD46-positive cell lines (HeLa, HeLa-CEA and Vero, FIG. 10A),expression of HXL with F induced extensive syncytia formation, to asimilar degree as unmodified H. The HXS protein supported syncytiaformation to a lower level, while no cell-cell fusion was observed incells expressing HX0. No significant difference was observed in thenumbers of syncytia in HeLa versus HeLa-CEA cells expressing any of thechimeric proteins. Thus addition of the long linker form of the scAb didnot impair MV H-induced cell-cell fusion via CD46. To assess thecontribution of the scAb-CEA interaction in the fusion process, weanalysed CEA-dependent syncytia formation in a CD46-negative background.For this we used MC38-CEA cells, a mouse cell line stably expressinghigh levels of cell surface human CEA (Robbins, et al. supra.), andtheir CEA-negative parent, MC38. FACS analysis (FIG. 10B) demonstratedlevels of CD46 to be similarly high on Vero, HeLa and HeLa-CEA cells,and undetectable on MC38 and MC38-CEA cells.

As expected, expression of CEA was high only on HeLa-CEA and MC38-CEAcells. Fusion was thus compared in MC38, MC38-CEA and Vero cells (FIGS.10C and 10D). In MC38 and MC38-CEA cells, co-expression of F withunmodified H induced a low level of syncytia formation, presumablyreflecting an inefficient, CD46-independent fusion mechanism for MV H.Strikingly, co-expression of F with chimeric HXL in MC38-CEA cells ledto extensive syncytia formation. Although at a reduced level comparedwith that in Vero cells, numbers of syncytia were over 100-fold greaterthan that seen with unmodified H. The HXS protein supported a reducedlevel of syncytia formation in both Vero and MC38-CEA cells, whileco-expression of HX0 with F yielded no detectable cell-cell fusion inany cell line. Thus MV H displaying the long linker form of the scAbinitiated cell-cell fusion via a novel receptor.

H. Recovery of Replication Competent MV Containing Chimeric HXL Proteinin Place of H

The ability of chimeric HXL to functionally replace unmodified H in thecontext of replicating virus was assessed. In a full length infectiousMV Edmonston cDNA, the H gene was replaced with that encoding HXL and,using the MV recovery protocol (Radecke, et al. supra)), virus wasisolated from individual syncytia formed in Vero cells.

The authenticity of the recovered MV-HXL virus was confirmed by Westernblot analysis of purified particles (FIG. 11A). Consistent with thesizes of transiently expressed HXL and H proteins (FIG. 8B), purifiedMV-HXL particles expressed an H protein of ˜110 kD, in contrast to thatof ˜80 kD expressed from unmodified MV. Furthermore, treatment ofpurified MV-HXL virions with Factor Xa protease demonstrated specificcleavage of the appended scAb, generating an 80 kD protein correspondingto unmodified H (FIG. 11B). As expected, Factor Xa treatment ofunmodified MV did not affect the size of the antigenic materialdetectable as H.

1. MV-HXL Virus Binds to the Surface of CD46-Positive and CD46-Negative,CEA-Positive Cells

The ability of MV and MV-HXL to bind cells expressing either CD46 or CEAat the surface was next compared by flow cytometry (FIG. 11C). Neithervirus was able to bind the surface of CD46-negative, CEA-negative MC38cells. In contrast, both viruses bound CD46-positive, CEA-negative Verocells, with unmodified MV demonstrating a slightly higher bindingability. Thus the addition of the scAb did not negate the interaction ofMV-HXL with cell surface CD46, consistent with the ability of HXLprotein to induce cell-cell fusion in CD46-positive cells. Importantly,the MV-HXL virus bound the surface of the CD46-negative, CEA-positiveMC38-CEA cell line, while binding of unmodified MV was negligible.

J. MV-HXL Virus Replicates in CEA-Positive Cells in the Absence of CD46

The infectivities of MV and MV-HXL for Vero, MC38 and MC38-CEA byobserving syncytia formation in the inoculated cells (FIG. 9A).Consistent with previous results, Vero cells were infectable by eithervirus. Significantly, infection of MC38-CEA cells with MV-HXL resultedin extensive syncytia formation. In contrast, infection of MC38-CEAcells with unmodified MV, and MC38 cells with either virus wasundetectable.

The replicative ability of MV-HXL was compared with that of unmodifiedMV by determining the viral titers achieved in Vero, HeLa, HeLa-CEA,MC38 and MC38-CEA cells by TCID50 assays using each of these cell linesas targets (FIG. 9B shows one typical example). MV-HXL replicated totiters almost indistinguishable from those obtained with unmodified MVin all three CD46-positive cell lines tested (7×105-5×107 pfu/mldepending on the cell line). Thus the ability of MV-HXL to replicate ina CD46-dependent manner was not affected by display of the scAb,consistent with our previous data.

Remarkably, MV-HXL reached a similar titer on CD46-negative,CEA-positive MC38-CEA cells as on CD46-positive cells (from independentexperiments, an average of 6.2×105+/−1.3×10⁵ pfu/ml), demonstrating thatits ability to replicate in a CEA-dependent manner was about asefficient as its CD46-dependent replication. Negligible infection(titers of <10² pfu/ml) was detected in MC38-CEA cells with unmodifiedMV, and in CEA-negative MC38 cells with either virus.

The infectivities of MV and MV-HXL were measured in all cell lines byinfecting each with an MOI of 3 and quantifying cell-associated virus byTCID50 titration 72 hours post-infection using Vero cells as targets.Since no intrinsic differences in replication of the two viruses in Verocells existed (FIG. 9B), no bias would be introduced using thisapproach. This method gave a reproducibly similar pattern of virustiters as the previous assay, but the absolute values were 1-2 logslower, with the titer of MV-HXL on MC38-CEA cells reaching3.1×10⁴+/−5.1×10³ pfu/ml (data not shown). This assay was used forsubsequent blocking experiments.

K. Replication of MV-Hxl in CD46-Negative, CEA-Positive Cells Depends ona Specific Interaction Between the Displayed ScAb And CEA

The infectivity of MV and MV-HXL for MC38-CEA and Vero cells followingincubation of virus with Factor Xa protease to cleave the displayedscAb, or pretreatment of cells with an αCEA mAb (COL1) to block cellsurface CEA, was examined. In both cases, virus was quantified from thecells 72 hours post-infection by TCID50 titration using Vero cells astargets (FIG. 12).

On Vero cells, neither treatment significantly affected the titer ofeither MV or MV-HXL; similarly, on MC38-CEA cells, the titer ofunmodified MV was unaffected by either treatment. Strikingly, cleavageof the displayed scAb by Factor Xa protease reduced the titer of MV-HXLon MC38-CEA cells by greater than 100-fold (from independentexperiments, an average of 177-fold +/−2-fold). Inhibition of MV-HXL bypretreating MC38-CEA cells with the

CEA mAb COL1 was less drastic but still significant, with an inhibitionof greater than 10-fold. These data confirm that the ability of MV-HXLto infect CEA-positive cells independently of CD46 depends on a specificinteraction between the displayed scAb and the targeted antigen.

Redirecting MV to a more clinically relevant target, CEA, establishes aprecedent for the development of useful MV-based therapeutics. Moreover,given the common features of scAb structure, the ability to display onescAb suggests that MV H may tolerate the addition of many scAbs asC-terminal fusions; a property highly desirable for targeting vectors.Remarkably, the presence of two independently folding domains in theseHαCEA molecules did not affect intracellular stability, suggesting theircorrect folding (Plemper, et al. supra.). More evidence that theconformation of the underlying H molecule was unaffected came from theability of HXL to dimerize with itself and with unmodified H and fromthe similar cell surface expression of all HαCEA proteins compared withthat of unmodified MV H.

Significantly, MV H displaying the long linker form of the αCEA scAb wasable to initiate efficient cell-cell fusion in cells expressing CD46,and also in CEA-positive cells independently of CD46. In contrast,addition of the zero and short forms of the scAb ablated or reduced thefusogenicity of the molecule, respectively. Since the scAbs of the HX0and HXS proteins may be oligomeric, they may sterically prevent fusioneither by blocking interaction with either cellular receptor or a secondfactor, or by a more complex interference in the oligomerisation of H orthe interaction between H and F oligomers. The HXL protein, however, ispredicted to display a monomeric scAb, which apparently does notobstruct any essential complex formations.

The two appended Ig-like domains to the H protein did not appear tointerfere with efficient particle assembly, despite the increase inmolecular mass of the H protein by 40%. Furthermore, the displayed scAbdid not impair entry and replication competence in CD46-positive cellsand significantly, enabled infection of cells lacking all human proteinsother than the targeted receptor, human CEA, to titers similar to thoseachieved on CD46-positive cells. Moreover, the CD46-independentinfectivity of MV-HXL for cells expressing CEA relied on a specific andinhibitable interaction between the scAb displayed on the virus and CEA.

CEA-dependent infection by MV-HXL can be described as ‘positiveretargeting’ of MV, since binding to CEA is followed by CEA-dependententry. These findings suggest the general applicability of MV as avector which can be positively retargeted to many other cell surfaceantigens by display of appropriate scAbs. In addition to its inherentcytotoxicity, developing retargeted vectors based on the safe andeffective MV-Edmonston vaccine strain supports the therapeutic use ofreplication competent virus. The combination of potent syncytiuminduction and replication competence may culminate in a more extensivespread amongst the target cell population.

EXAMPLE 8 Attenuated Measles Virus Targeting Specificity for EGF or IGF1

Hybrid proteins consisting of the epidermal growth factor (EGF) or theinsulin-like growth factor-1 (IGF1) linked to the extracellular(carboxyl) terminus of the MV-Edm attachment protein hemagglutinin (H)were produced. The standard H protein gene was replaced by one codingfor H/EGF or H/IGF1 in cDNA copies of the MV genome. Recombinant viruseswere rescued and replicated to titers approaching those of the parentalstrain. MV displaying EGF or IGF1 efficiently entered CD46 negativerodent cells expressing the human EGF or the IGF1 receptor, respectivelyand the EGF-virus caused extensive syncytia formation, and cell death.Taking advantage of a factor Xa protease recognition site engineered inthe hybrid H proteins, the displayed domain was cleaved off from virusparticles, and specific entry in rodent cells was abrogated.

A. Plasmids

The parental plasmids pCG-F and pCG-H code for the F and H proteins ofMV-Edmonston (9). Plasmids pCG-H/SfiI/NotI and pCG-H/XSfiI/NotI, thesecond including a factor Xa protease (FXa) cleavage signal before theSfiI/NotI cloning sites, were constructed and digested with SfiI andNotI to provide the backbone in which the coding regions for thedisplayed domains were inserted. The constructs pCG-H/hEGF, pCG-H/XhEGF,pCG-H/hIGF1 and pCG-H/XhIGF1 were made by transferring the SfiI/NotIhEGF and hIGF1 fragments from pEGF-GS1A1 (Peng, k. W., 1997, Thesis.University of Cambridge)) and pIGFNA1 (Chadwick, et al. J. Mol. Biol.285: 485-494), respectively, into SfiI/NotI-digested pCG-H/SfiI/NotI andpCG-H/XSfiI/NotI. The coding sequence of the linker region (IEGRAAQPAMA,one letter code) is 5′-ATCGAGGGAAGGGCGGCCCAGCCGGCCATGGCC-3′. The fourconstructs were tested to verify their functionality in cell fusionassays.

The PacI-SpeI fragments containing the hybrid H genes were corrected tocomply to the rule of six (7(Calain, et al. J. Virol. 67: 4822-4830,1993)) by a PCR deleting one nucleotide between the stop codon(underlined) and the SpeI site (italics), the final sequence being5′-TAGTAACTAGT. The fragments were then inserted into PacI-SpeI digestedp(+)MV-NSe (Singh, et al. J. Virol. 73: 4823-4828, 1999) encoding the MVEdmonston antigenome, yielding plasmids p(+)MV-H/hEGF, p(+)MV-H/XhEGF,p(+)MV-H/hIGF1 and p(+)MV-H/XhIGF1.

Plasmids p(+)MVgreen-H/XhEGF and p(+)MVgreen-H/XhIGF1 encoding theenhanced green fluorescent protein as an additional transcription unitupstream of the N gene (Hangartner, 1997. M.Sc. Thesis. University ofZurich) were constructed using a unique SacII restriction site locatedwithin the P gene, and the SpeI site found downstream of the H codingregion. The SacII/SpeI fragments from p(+)MV-H/XhEGF and p(+)MV-H/XhIGF1containing the hybrid H genes were inserted into the SacII/SpeI openedbackbone of p(+)MVgreen (Duprex, J. Virol. 73: 9568-9575, 1999)).

Construction of PCG-H/EGF and PCG-H/IGF.

Unmodified measles virus (MV) F and H proteins were encoded by theexpression plasmids pCG-F and pCG-H, respectively (Cathomen et al.,1995, Virology, supra). To make the chimeric MV H expression constructs,the SfiI site in pCG-H was first deleted so that the displayed ligandscould subsequently be introduced as SfiI/NotI fragments. This wasachieved by digesting pCG-H with SfiI, filling in the cohesive ends bytreating with the Klenow fragment of E. coli DNA polymerase and dNTPs,then re-ligating the purified, blunt-ended product. This construct,termed pCG-H(SfiI-) was tested to verify its functionality in cellfusion assays.

To introduce the SfiI/NotI cloning site at the C-terminus of the Hsequence, thus enabling ligands to be inserted as SfiI/NotI fragments,oligonucleotides HXmabak (5′-CCG GGA AGA TGG AAC CAA TGC GGC CCA GCC GGCCTC AGG TTC AGC GGC CGC ATA GTA GA-3′) and HSpefor (5′-CTA GTC TAC TATGCG GCC GCT GAA CCT GAG GCC GGC TGG GCC GCA TTG GTT CCA TCT TC-3′) weresynthesised. When annealed, these two oligonucleotides form a DNAfragment with XmaI and SpeI cohesive ends which was ligated withXmaI/SpeI digested pCG-H(SfiI-) backbone. The correct sequence of thisconstruct, pCG-H-SfiI/NotI was verified by DNA sequencing.

To make the construct pCG-H-FX-SfiI/NotI, which includes a factor Xaprotease (FX) cleavage signal before the SfiI/NotI cloning sites at theC-terminus of the MV H sequence, oligonucleotides HXmaFXbak (5′-CCG GGAAGA TGG AAC CAA TAT CGA GGG AAG GGC GGC CCA GCC GGC CTC AGG TTC AGC-3′)and HNotFXfor (5′-GGC CGC TGA ACC TGA GGC CGG CTG GGC CGC CCT TCC CTCGAT ATT GGT TCC ATC TTC-3′) were synthesised. When annealed, these twooligonucleotides form a DNA fragment with XmaI and NotI cohesive endswhich was ligated with the XmaI/NotI digested pCG-H-SfiI/NotI backbone.The correct sequence of this construct was verified by DNA sequencing.

Constructs pCG-H-EGF, pCG-H—X-EGF, pCG-H-IGF and pCG-H—X-IGF were madeby transferring the SfiI/NotI EGF and IGF fragments from pEGF-GS1A1(Peng, PhD thesis, the entierty of which is incorporated by referenceherein) and pIGFA1 (WO97/03357, Russell et al., the entierty of which isincorporated by reference herein) respectively into SfiI/NotI digestedpCG-H-SfiI/NotI and pCG-H-FX-SfiI/NotI. FIG. 14A shows the pCG-H/(X)hEGFand pCG-H/(X)hIGF1 constructs.

B. Cells, Viruses and Infections

Cells were grown in Dulbecco's modified Eagle's medium supplemented with5% fetal calf serum (DMEM-5) for Vero (African green monkey kidney) andA431 (human carcinoma) cells, 10% fetal calf serum (DMEM-10) for CHO(Chinese hamster ovary) cells, or DMEM-10 containing 1 mg/ml G418 forthe rescue helper cell line 293-3-46 (human embryonic kidney),CHO-hEGFr, CHO-hEGF.tr and 3T3-hIGF1r (mouse fibroblasts) cells. TheEdmonston B-based parental MV strain and all its recombinant derivativeswere rescued, propagated and purified basically as described previously(Radecke, et al. supra.)). Viral titers were determined by 50% end pointdilution assays (Cathomen, et al., J. Virol 72: 1224-1234, 1998).

Infections were generally performed in Opti-MEM I reduced serum medium(O-MEM, Gibco). One day prior to infection, 4×10⁵ cells were seeded intoa 35-mm well. Before infection, cells were washed with 2 ml O-MEM andthe infection was performed in 1 ml O-MEM for 1-2 hours at 37° C. Thecells were washed again in 2 ml O-MEM and overlaid with 2 ml DMEM-5 orDMEM-10 depending on the cell line infected.

C. One-Step Growth Analysis

Vero cells (5×10⁵) were infected with parental and recombinant MV at anMOI. of 3 for 2 hours. After infection, the cells were overlaid with 1ml DMEM-10 and incubation was continued at 32° C. Released andcell-associated virus samples were collected at different times postinfection. Cell-associated virus was subjected to one cycle offreeze/thawing and then the samples were stored at −80° C. for at least4 hours prior to titration. Before titration, the samples werecentrifuged in a table-top centrifuge (Spectrafuge 16M, Labnet) for 2min at 8000 rpm to remove cell debris.

D. Purification of Viral Particles

For each virus two T175 bottles (Falcon) with Vero cells of 80%confluency (2×10⁷) were infected at an MOI of 0.1 for 2 hours at 37° C.The infection solution was replaced by 20 ml DMEM-5 and cells wereincubated at 32° C. until 80-100% of all nuclei were found in syncytia.Supernatants were collected in 36-ml polypropylene tubes (Sorvall) andclarified by 30 min centrifugation at 8000 rpm in a Surespin 630 rotor(Sorvall). Clarified supernatants were transferred into new 36-ml tubesand pelleted by velocity centrifugation (2 hours at 28000 rpm) through20% sucrose onto a 60% sucrose cushion prepared in TNE buffer (10 mMTris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA [pH 7.5]). The fractioncontaining the virus was harvested, diluted by addition of TNE andpelleted at the bottom of the tube by 2 hours centrifugation at 28000rpm. After careful removal of all liquid, viruses were dissolved in 200μl phosphate-buffered saline (PBS, Gibco) and stored at −80° C.

E. Receptor Saturation Assays

Vero, CHO-hEGFr and 3T3-hIGFr cells (5×10⁵) were incubated in 0.5 mlO-MEM containing either hEGF or hIGF1 (R&D Systems, 236-EG and 291-G1)for 30 min at 37° C. For infection, another 0.5 ml O-MEM containing thevirus was added to each well, yielding final hEGF and hIGF1concentrations of 0.25 and 1 μM or 0.05 and 0.2 μM, respectively, in 1ml of O-MEM. Infection was allowed to continue for 3 hours at 37° C. Thecells were washed with 2 ml O-MEM and overlaid with DMEM-10 without hEGFor hIGF1.

F. Factor Xa Protease Pretreatment of Viral Particles

Virus stocks were diluted in O-MEM to a concentration of 10⁴ pfu in 20μl. Forty μl of diluted virus were incubated with either 0.2 μg or 2 μgFXa protease, yielding a concentration of 5 and 50 μg/ml FXa,respectively. After 1 hour incubation at 37° C., 2 ml O-MEM were addedto each virus sample. Cells were infected with 1 ml virus sample per 35mm well as described above.

G. Immunoblotting

Approximately 5000 plaque-forming units (pfu) of purified virus in 10 μlof PBS were lysed by addition of 9 μl lysis buffer (50 mM Tris [pH 8.0],62.5 mM EDTA, 1% IGEPAL CA-630 (former NP-40), 0.4% deoxycholate). Lysedvirus samples were adjusted to a volume of 20 μl with either 1 μl of PBSor with 1 μl of a 1 μg/μl FXa protease solution (New England Biolabs),incubated for 1 hour at room temperature and subjected to sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).

The gels were blotted onto polyvinylidene difluoride membranes(Millipore). The membranes were blocked with 5% bovine serum albumin, 5%skim milk powder in TBST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween20) for 1.5 hours at room temperature. They were then incubated witheither goat anti-MV antiserum diluted 1:1000 (courtesy of S. Udem) orrabbit anti-H cytoplasmic tail antiserum diluted 1:2500 (10). Afterintense washing the proteins were visualized by incubation withperoxidase-conjugated goat anti-rabbit IgG (Jackson Immuno Research,111-035-003) and peroxidase-conjugated rabbit anti-goat IgG (Calbiochem,401504), respectively, for 1 hour at room temperature and subsequenttreatment with chemiluminescent substrate (Pierce, 34080ZZ).

H. ELISA

ELISA plates were coated with 100 μl of 1 μg/ml dilutions of monoclonalanti-hEGF and anti-hIGF1 antibodies (R&D Systems, MAB236 and MAB291) for2 hours at 37° C. and blocked by incubation with 200 μl of 1% blockingreagent (Boehringer Mannheim, 1 096 176) in TBS (10 mM Tris [pH 8.0],150 mM NaCl). The plates were incubated with 100 μl recombinant MVdiluted in 1% blocking solution overnight at 4° C.

Plates were washed three times with 200 μl TBS and bound virus wasdetected by incubation with 100 μl of a rabbit anti-Hcterm specificantiserum diluted 1:100 in 1% blocking solution for 2 hours at 4° C. TheHcterm antiserum was raised in rabbits against a peptide correspondingto the 12H-protein carboxy-terminal amino acids (NH2-CTVTREDGTNRR)linked to keyhole limpet hemocyanin through the naturally occurringcysteine (C). For detection, peroxidase-conjugated goat anti-rabbit IgG(Jackson Immuno Research, 111-035-003) diluted 1:5000 in 1% blockingsolution was added for 1 hour at 4° C. and after intense washing thecolor reaction was performed using the POD substrate from BoehringerMannheim (1 363 727).

I. FACScan Analysis

Expression levels of CD46, hEGFr and hIGR1r were determined byinoculating 5×105 cells in 50 μl PBS with 1:100 dilutions of monoclonalanti-CD46 clone 11/88 (courtesy of J. Schneider-Schaulies), monoclonalanti-hEGFr clone 528 (Santa Cruz, sc-120) and monoclonal anti-hIGF1rclone 33255.111 (R&D Systems, MAB391) for 1 hour on ice. After washing,the cells were incubated with a 1:50 dilution of afluorescein-conjugated donkey anti-mouse IgG (Jackson Immuno Research,715-095-151) for 30 min on ice, washed again, fixed in PBS containing 1%paraformaldehyde and analyzed.

To analyze virus binding, 105 CHO-hEGFr and 3T3-hIGF1r cells in a totalvolume of 50 μl PBS were incubated with 104 pfu of purified Edmonston orrecombinant MV for 2 hours on ice. The samples were washed once in 3.5ml PBS with 2% FCS and incubated in 50 μl PBS containing a 1:100dilution of the monoclonal anti-H antibody I41 (Sheshberadaran, et al.Virology 128: 341-353, 1983) for 1.5 hours on ice. Subsequentincubations were performed as described above.

J. H Proteins Displaying a Specificity Domain Functionally ReplaceStandard H Protein

The human epidermal growth factor (hEGF, 53 amino acids) were used togenerate a hybrid protein in which the hEGF coding region was fused inframe with the H coding region, but eliminating the last two arginineresidues, to avoid the possibility of introducing an undesired furincleavage site (H/hEGF, FIG. 14A, second line from bottom). A flexiblelinker region (AAQPAMA) was added between the domains to increase theprobability of independent folding function. In another embodiment, thehuman insulin-like growth factor-1 (hIGF1, 70 amino acids) was fused tothe H protein. A factor Xa protease cleavage site (IEGR) was addedbefore the linker region (H/XhEGF, FIG. 14A, second line from bottom).Hybrid H/hIGF1 and H/XhIGF1 proteins were constructed (FIG. 14A, bottomline). When the hybrid proteins were co-expressed with a MV F protein,H/hEGF and H/XhEGF retained the same level of fusogenicity as parentalH, whereas fusogenicity of H/hIGF1 and H/XhIGF1 was reduced but remainedclearly over background.

The hybrid H proteins functionally substitute for H in viral particles.In one experiment, the H gene of an infectious MV cDNA was cloned inp(+)MV-Nse(Singh, et al. J. Virol 73: 4823-4828) with genes coding forthe hybrid proteins. Helper cells (Radecke, et al. supra) weretransfected with those plasmids. Three to four days after transfectionwith p(+)MV-NSe, p(+)MV-H/hEGF and p(+)MV-H/XhEGF, and five to sevendays after transfection with p(+)MV-H/hIGF and p(+)MV-H/XhIGF, syncytiawere detected, indicating virus rescue. Infectivity was passaged in Verocells, the African green monkey cell line routinely used to grow andtitrate MV, and the recombinant viruses reached titers in a rangesimilar to that of the parental virus (see below).

As shown in FIG. 14B, purified particles from the MV-H/hEGF, MV-H/XhEGFand MV-H/XhIGF1 viruses contained a similar amount of H protein asparental MV, but more N and M proteins. These results suggest that thehybrid H proteins are incorporated slightly less efficiently than H inparticles, and that the particle to infectivity ratio of the recombinantMV is slightly higher than for the parental strain.

The specific proteolytic cleavage of the displayed domain from purifiedvirus particles occurred. In FIG. 14C, it is shown that factor Xa (FXa)protease specifically cleaves off the displayed domain from the Hprotein of MV-H/XhEGF (lane 6) and MV-H/XhIGF1 (lane 8), but not fromthe control viruses MV (lane 2) and MV-H/hEGF (lane 4).

The levels of expression of CD46 and of the targeted receptors in Verocells was determined by FACs analysis, as shown in FIG. 14D. The EGFreceptor (hEGFr, continuous line), IGF receptor (hIGF1r, dotted line),and CD46 (interrupted line) were all expressed. For one step growthanalysis Vero cells were infected in parallel with a MOI of 3 of eitherMV-H/XhEGF, MV-H/XhIGF1, or the parental MV strain. Released andcell-associated virus were harvested at 12 hour intervals and titratedon Vero cells.

Results spanning the times from 24 to 60 hours post-infection are shownin FIG. 14E. Cell-associated titers of MV-H/XhEGF (squares) rose slowerthan those of the parental strain (diamonds), whereas those ofMV-H/XhIGF1 (triangles) were intermediate, but all viruses reachedsimilar maximum titers of about 5×106/ml (FIG. 14E, left panel). Releaseof infectious virus in the supernatant followed slower kinetics, asexpected and peaked at 1.7×106 for the parental strain, and at 3.5 and6.3 times lower levels (FIG. 14E, left panel), for MV-H/XhIGF1 andMV-H/XhEGF, respectively. These results indicate that in Vero cellsreplication and intracellular assembly of the two recombinant viruses isslower than that of the parental strain. Nevertheless, the maximumtiters of cell-associated virus are similar for all three strains.Release of the two recombinant viruses is slightly less efficient thanthat of parental MV.

The displayed domains are accessible for binding by antibodies. FIG. 15Ashows that plates coated with an anti-hEGF monoclonal antibody incubatedwith increasing amounts of MV-H/XhEGF retained increasing amounts ofvirus (left panel, white columns), whereas on plates coated withanti-hIGF1 only background retention levels were detected (right panel,white columns). The opposite was the case for the MV-H/XhIGF1 virus,which was selectively retained by the anti-hIGF1 monoclonal antibody(black columns on right panel).

K. MV Displaying a Specificity Domain Bind to, Infect, and Fuse, RodentCells Expressing the Cognate Receptor.

Edmonston MV-derived strains are expected to bind CD46, which isexpressed on the surface of all cell types from most primates. To verifyif the displayed hEGF and hIGF1 domains did confer attachment to theircognate receptors, we relied on CD46 negative rodent cells: Chinesehamster ovary cells stable expressing the hEGF receptor and mouseNIH-3T3 cells stable expressing the hIGF1 receptor 3T3-hIGF1r (Lammers,et al. EMBO 8: 1369-1375).

FIG. 15B presents a fluorescence-activated cell sorter (FACS) analysisconfirming that CHO-hEGFr cells (upper left panel) and 3T3-hIGF1r (upperright panel) express the corresponding human protein on their surface.When these cells were incubated with MV-H/XhEGF (lower left panel) orMV-H/XhIGF1 (lower right panel), respectively, and virus binding wasdetected with an anti-H monoclonal antibody (thick line), a shift in thenumber of strongly fluorescent cells from background antibody binding(gray area) and from binding of control MV (thin lines) was shown inboth cell lines.

To detect viral gene expression early after entry, viruses expressinghigh levels of the enhanced green fluorescent protein (eGFP) wereconstructed (5Yang, et al. NAR 24: 4592-4593, 1996). For this, a MVgenomic plasmid containing an eGFP transcription unit (Duprex, et al.supra) was combined with p(+)MV-H/XhEGF and p(+)MV-H/XhIGF1. The newviruses were rescued and named MVgreen-H/XhEGF and MVgreen-H/XhIGF1.

FIGS. 16A-J present three time points (24, 48 and 72 hours postinfection, p.i.) of an infection of CHO-hEGFr cells with MVgreen-H/XhEGFat a multiplicity of infection (m.o.i.) of 1. At 24 hours p.i. (panel A)fluorescence was detected in single cells; at 48 hours p.i. (panel B) alarge fraction of the cells showed a strong signal and, remarkably,several fused cells were registered; at 72 hours p.i. the majority ofthe cells had fused in large syncytia (panel D) which were fluorescent(panel C). In controls CHO-hEGFr cells infected with MVgreen nofluorescence was detected 24 hours p.i. and few positive cells 72 hoursp.i. (panel E). The same was true for another negative control, CHOcells infected with MVgreen-H/XhEGF (panel I shown 72 hours afterinfection).

The few small syncytia detected in MVgreen infected CHO-hEGFr cells andMVgreen-H/XhEGF infected CHO cells (panel F and J) were not fluorescent(panels E and I, respectively) and thus were due to spontaneous fusionat high cell density. On the other hand CHO-hEGFr.tr cells, expressing ahEGF receptor with a truncated cytoplasmic tail supported efficientMVgreen-H/XhEGF infection, which resulted in cell fusion (panels G andH, shown 72 hours after infection). Thus, CHO cells expressing the humanEGF receptor, or a mutant receptor without the cytoplasmic tail, supportefficient, CD46-independent cell entry of MVgreen-H/XhEGF, indicatingthat internalization or intracellular signaling do not affect MV entry.Strikingly, in these cells the MV infection results in extensivesyncytia formation.

FIGS. 17A-D present 3T3-hIGF1r cells 24 hours after infection with a MOIof 3 of MVgreen-H/XhIGF1 (panels A and B) and MVgreen (panels C and D),respectively. Many cells infected with MVgreen-H/XhIGF1 were stronglyfluorescent (panel A), whereas only rare fluorescent cells were detectedafter MVgreen infection (panel C). To gain some information on therelative efficiency of entry of MVgreen-H/XhIGF1 and MVgreen in3T3-hIGF1r cells, these cells were infected with either one of theviruses at a MOI of 3, 0.3 or 0.03. In a 35 mm dish, 735 positive cellsinfected with MVgreen-H/XhIGF1 at an MOI of 0.03 were counted, and 87positive cells infected with MVgreen at an MOI of 3. Thus, about 1000times more infectious particles of MVgreen than of MVgreen-H/XhIGF1 wereneeded to elicit detectable eGFP expression in the same number of3T3-hIGF1r cells. Even late in infection of 3T3-hIGF1r cells withMVgreen-H/XhIGF1 fusion was limited, in line with the limited fusion ofprimate Vero cells (data not shown).

The interaction of the displayed domains with their receptor mediatesvirus entry. As shown in FIG. 18A, top panel, black columns, theaddition of 0.25 or 1 μM soluble human EGF to the medium of CHO-hEGFrcells reduced the number of cells infected with MVgreen-H/XhEGFapproximately 5-6 times. There was no inhibition of the MVgreeninfection of Vero cells by 1 μM soluble EGF (FIG. 18A, top panel, graycolumns), but interestingly there was a small but reproducible effect ofsoluble EGF in inhibiting the MVgreen-H/XhEGF infection of those cells(FIG. 18A, top panel, white columns). Analogously, soluble human IGFinterfered selectively with the infection of MVgreen-H/XhIGF1, morestrongly on 3T3-hIGFr cells than on Vero cells (FIG. 18A, lower panel).These results indicated that entry of MVgreen-H/XhEGF orMVgreen-H/XhIGF1 could be competed by the addition of a soluble form ofhEGF or hIGF1, respectively to the medium.

When MV-H/XhEGF particles were treated with 5 or 50 μg/ml of factor Xaprotease for one hour, the number of green fluorescent CHO-hEGFr cellsdiminished by almost two orders of magnitude (FIG. 18B, left panel,black column), whereas factor Xa treatment did not significantly changethe infectivity of these particles on Vero cells (FIG. 18B, left panel,white columns). Analogously, proteolytic cleavage of MV-H/XhIGF1 virusparticles with factor Xa protease resulted in loss of more than 80% ofthe infectivity selectively in 3T3-hIGF1r cells (FIG. 18B, right panel,compare black and white columns). Thus, by two different approaches,competition with a soluble form of the displayed domain, and proteolyticcleavage of that domain from viral particles, it was confirmed thatentry of recombinant MV in rodent cells depends on the interaction ofthe specificity domain with the cognate receptor.

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing detailed description is providedfor clarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims.

1-26. (canceled)
 27. A method for delivering a Paramyxoviridae virus toa tumor in a mammal, said method comprises administering saidParamyxoviridae virus to said mammal under conditions wherein saidParamyxoviridae virus enters a cell in said tumor, wherein saidParamyxoviridae virus comprises a polypeptide comprising aParamyxoviridae virus polypeptide sequence and a heterologous sequence,wherein said heterologous sequence binds to a receptor present on saidcell.
 28. The method of claim 27, wherein said Paramyxoviridae virus isa measles virus.
 29. The method of claim 27, wherein said heterologoussequence is fused via an intervening amino acid linker to saidParamyxoviridae virus polypeptide sequence.
 30. The method of claim 27,wherein said Paramyxoviridae virus polypeptide sequence comprises anamino acid sequence of a Paramyxoviridae virus F or H protein.
 31. Themethod of claim 27, wherein said heterologous sequence is a single chainantibody.
 32. The method of claim 31, wherein said single chain antibodybinds a carcinoembryonic antigen.
 33. The method of claim 31, whereinsaid single chain antibody binds a CD38 polypeptide.
 34. The method ofclaim 27, wherein said heterologous sequence is an EGF polypeptide. 35.The method of claim 27, wherein said heterologous sequence is an IGF-1polypeptide.
 36. The method of claim 27, wherein said receptor is acarcinoembryonic antigen, a CD38 polypeptide, an EGF receptorpolypeptide, or an IGF-1 receptor polypeptide.
 37. A Paramyxoviridaevirus comprising a polypeptide comprising a Paramyxoviridae viruspolypeptide sequence and a heterologous sequence, wherein saidheterologous sequence binds to a receptor present on a cell in a tumor.38. The Paramyxoviridae virus of claim 27, wherein said Paramyxoviridaevirus is a measles virus.
 39. The Paramyxoviridae virus of claim 27,wherein said heterologous sequence is fused via an intervening aminoacid linker to said Paramyxoviridae virus polypeptide sequence.
 40. TheParamyxoviridae virus of claim 27, wherein said Paramyxoviridae viruspolypeptide sequence comprises an amino acid sequence of aParamyxoviridae virus F or H protein.
 41. The Paramyxoviridae virus ofclaim 27, wherein said heterologous sequence is a single chain antibody.42. The Paramyxoviridae virus of claim 41, wherein said single chainantibody binds a carcinoembryonic antigen.
 43. The Paramyxoviridae virusof claim 41, wherein said single chain antibody binds a CD38polypeptide.
 44. The Paramyxoviridae virus of claim 27, wherein saidheterologous sequence is an EGF polypeptide.
 45. The Paramyxoviridaevirus of claim 27, wherein said heterologous sequence is an IGF-1polypeptide.
 46. The Paramyxoviridae virus of claim 27, wherein saidreceptor is a carcinoembryonic antigen, a CD38 polypeptide, an EGFreceptor polypeptide, or an IGF-1 receptor polypeptide.